Dgat genes for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

ABSTRACT

Polynucleotide sequences encoding diacylglycerol acyltransferase genes and the use of these acyltransferases for increased seed storage lipid production and altered fatty acid profiles in oilseed plants are disclosed. Transgenic soybean seed having increased total fatty acid content of at least 20% and altered fatty acids when compared to the total fatty acid content of non-transgenic, null segregant soybean seed are described. Methods for increasing the total fatty acid content of a soybean seed by at least 20% include steps of transformation and selection.

This application is a continuation of U.S. application Ser. No.15/402,540, filed Jan. 10, 2017, now allowed, which is a continuation ofU.S. application Ser. No. 14/475,790, filed Sep. 3, 2014, now U.S. Pat.No. 9,574,207, which is a continuation of U.S. application Ser. No.13/295,316, filed Nov. 14, 2011, now U.S. Pat. No. 8,829,273, which is acontinuation of U.S. application Ser. No. 12/470,509, filed May 22,2009, now U.S. Pat. No. 8,143,476, which claims the benefit of U.S.Provisional Application No. 61/055,580, filed May 23, 2008, now expired,the entire contents of which are hereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file nameBB1644USCNT3_SeqLst.txt created on Aug. 30, 2018 and having a size of1312 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention is in the field of biotechnology, in particular, thispertains to polynucleotide sequences encoding diacylglycerolacyltransferase genes and the use of these acyltransferases forincreased seed storage lipid production and altered fatty acid profilesin oilseed plants.

BACKGROUND OF THE INVENTION

Plant lipids have a variety of industrial and nutritional uses and arecentral to plant membrane function and climatic adaptation. These lipidsrepresent a vast array of chemical structures, and these structuresdetermine the physiological and industrial properties of the lipid. Manyof these structures result either directly or indirectly from metabolicprocesses that alter the degree of unsaturation of the lipid. Differentmetabolic regimes in different plants produce these altered lipids, andeither domestication of exotic plant species or modification ofagronomically adapted species is usually required to produceeconomically large amounts of the desired lipid.

There are serious limitations to using mutagenesis to alter fatty acidcomposition and content. Screens will rarely uncover mutations that a)result in a dominant (“gain-of-function”) phenotype, b) are in genesthat are essential for plant growth, and c) are in an enzyme that is notrate-limiting and that is encoded by more than one gene. In cases wheredesired phenotypes are available in mutant corn lines, theirintrogression into elite lines by traditional breeding techniques isslow and expensive, since the desired oil compositions are likely theresult of several recessive genes.

Recent molecular and cellular biology techniques offer the potential forovercoming some of the limitations of the mutagenesis approach,including the need for extensive breeding. Some of the particularlyuseful technologies are seed-specific expression of foreign genes intransgenic plants [see Goldberg et al (1989) Cell 56:149-160], and theuse of antisense RNA to inhibit plant target genes in a dominant andtissue-specific manner [see van der Krol et al (1988) Gene 72:45-50].Other advances include the transfer of foreign genes into elitecommercial varieties of commercial oilcrops, such as soybean [Chee et al(1989) Plant Physiol. 91:1212-1218; Christou et al (1989) Proc. Natl.Acad. Sci. U.S.A. 86:7500-7504; Hinchee et al (1988) Bio/Technology6:915-922; EPO publication 0 301 749 A2], rapeseed [De Block et al(1989) Plant Physiol. 91:694-701], and sunflower [Everett et al (1987)Bio/Technology 5:1201-1204], and the use of genes as restrictionfragment length polymorphism (RFLP) markers in a breeding program, whichmakes introgression of recessive traits into elite lines rapid and lessexpensive [Tanksley et al (1989) Bio/Technology 7:257-264]. However,application of each of these technologies requires identification andisolation of commercially-important genes. Most free fatty acids becomeesterified to coenzyme A (CoA), to yield acyl-CoAs. These molecules arethen substrates for glycerolipid synthesis in the endoplasmic reticulumof the cell, where phosphatidic acid and diacylglycerol (DAG) areproduced. Either of these metabolic intermediates may be directed tomembrane phospholipids (e.g., phosphatidylglycerol,phosphatidylethanolamine, phosphatidylcholine) or DAG may be directed toform triacylglycerols (TAGs), the primary storage reserve of lipids ineukaryotic cells.

Diacylglycerol acyltransferase (“DGAT”) is an integral membrane proteinthat catalyzes the final enzymatic step in the production oftriacylglycerols in plants, fungi and mammals. This enzyme isresponsible for transferring an acyl group from acyl-coenzyme-A to thesn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol(“TAG”). DGAT is associated with membrane and lipid body fractions inplants and fungi, particularly, in oilseeds where it contributes to thestorage of carbon used as energy reserves. TAG is believed to be animportant chemical for storage of energy in cells. DGAT is known toregulate TAG structure an direct TAG synthesis. Furthermore, it is knownthat the DGAT reaction is specific for oil synthesis.

TAG is the primary component of vegetable oil in plants, It is used bythe seed as a stored form of energy to be used during seed germination.

Two different families of DGAT proteins have been identified. The firstfamily of DGAT proteins (“DGAT1”) is related to the acyl-coenzymeA:cholesterol acyltransferase (“ACAT”) and has been described in U.S.Pat. Nos. 6,100,077 and 6,344,548. A second family of DGAT proteins(“DGAT2”) is unrelated to the DGAT1 family and is described in PCTPatent Publication WO 2004/011671 published Feb. 5, 2004. Otherreferences to DGAT genes and their use in plants include PCT PublicationNos. WO2004/011,671, WO1998/055,631, and WO2000/001,713, and US PatentPublication No. 20030115632.

Applicants' Assignee's copending published patent application US2006-0094088 describes genes for DGATs of plants and fungi and their useis in modifying levels of polyunsaturated fatty acids (“PUFAs”) inedible oils.

Applicants' Assignee's published PCT application WO 2005/003322describes the cloning of phosphatidylcholine diacylglycerolacyltransferase and DGAT2 for altering PUFA and oil content inoleaginous yeast.

SUMMARY OF THE INVENTION

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 20% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

In a second embodiment, the present invention concerns a method forincreasing the total fatty acid content of a soybean seed comprising:

(a) transforming at least one soybean cell with one or more recombinantconstructs having at least one DGAT sequence and a constructdownregulating plastidic phosphoglucomutase activity wherein the DGATsequence and the plastidic phosphoglucomutase construct are on the sameor separate recombinant constructs;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 20% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

Any of the transgenic seed of the invention may comprise a recombinantconstruct having at least one DGAT sequence which can be selected fromthe group consisting of DGAT1, DGAT2 and DGAT1 in combination withDGAT2. Furthermore, the DGAT sequence can be a Yarrowia sequence.

Any of the transgenic seed of the invention may comprise a recombinantconstruct having downregulated plastidic phosphoglucomutase activity.

Also within the scope of the invention are product(s) and/orby-product(s), and progeny, obtained from the transgenic soybean seedsof the invention.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing, whichform a part of this application.

FIGS. 1A, 1B and 1C provide plasmid maps for pFBAIn-YLDGAT1, forpFBAIn-YLDGAT2, and for pFBAIn-MOD1, respectively.

FIGS. 2A and 2B provide plasmid maps for KS352 and KS332, respectively.

FIGS. 3A, 3B, and 3C provide plasmid maps for KS349, KS362, and KS364,respectively.

FIGS. 4A and 4B provide a strong correlation (R²≥0.59) between the oleicacid content and the total esterified fatty acid content for somaticembryos generated with KS349 compared to KS352.

FIGS. 5A, 5B, 5 c, and 5D provide a strong correlation (R²≥0.67) betweenthe oleic acid content and the total esterified fatty acid content forsomatic embryos generated with KS362 alone or in combination with KS349as well as with KS364.

FIGS. 6A and 6B provide a correlation (R²≥0.45) between the oleic acidcontent and the oil content for transgenic soy seed (T1 generation)generated by co-transformation of plasmids KS349 and KS362.

FIGS. 7A and 7B provide oil content and seed weight of T1 seed generatedby co-transformation of plasmids KS349 and KS362 (A) and KS362 alone(B).

FIGS. 8A and 8B provide hybridization results from genomic DNA blots.Genomic DNA was isolated from transgenic soybeans obtained from eventsAFS4818.1.2, AFS4818.1.3, AFS4818.1.5, AFS48182.6, AFS4818.1.9 (SeeExample 6). DNA was digested with EcoRI or HindIII and run out on a geland blotted to nylon filters [AFS4818.1.2 lanes 1 and 2, AFS4818.1.3lanes 3 and 4, AFS4818.1.5 lanes 5 and 6, AFS48182.6 lanes 7 and 8,AFS4818.1.9 lanes 9 and 10, and lanes 11 and 12 are non-transgenicwild-type DNA also digested with EcoRi and HindIII]. Hybridizationprobes were a Yarrowia DGAT1-specific probe for the upper blot (A) andthe lower blot was probed with a Yarrowia DGAT2 specific probe.

FIG. 9 provides hybridization results from genomic DNA blots. The blotsare similar to those described in FIGS. 8A and 8B except the DNAs wereall digested with BstXI and the blot was probed with a Yarrowia DGAT2specific probe.

The sequence descriptions summarize the Sequences Listing attachedhereto. The Sequence Listing contains one letter codes for nucleotidesequence characters and the single and three letter codes for aminoacids as defined in the IUPAC-IUB standards described in Nucleic AcidsResearch 13:3021-3030 (1985) and in the Biochemical Journal219(2):345-373 (1984).

Summary of Nucleic Acid and Protein SEQ ID Numbers

Nucleic acid Protein Description and Abbreviation SEQ ID NO. SEQ ID NO.Yarrowia lipolytica DGAT1 gene   1 (1581 bp) Plasmid pYDA1   2 (8325 bp)Plasmid py75   3 (7518 bp) Plasmid pY75 YLDGAT1:   4 YLDGAT1 insertedinto pY75 (9109) Plasmid pRS425   5 (6849 bp) Plasmid pGDP425   6 (7494bp) Yarrowia lipolytica DGAT2 gene   9 10 (1545 bp) (514aa) Plasmid pY75YLDGAT2, pY75  11 with YL DGAT2 inserted (9070 bp) Yarrowia lipolyticaDGAT1  16 gene variant with Ncol and (1603 bp) Notl sites added Yarrowialipolytica DGAT2  17 gene variant with Ncol and (1567 bp) Notl sitesadded Plasmid pFBAIN-MOD-1  18 (6991 bp) Plasmid pFBAIN-YLDGAT,  19pFBAIN with YL DGAT1 (8568 bp) inserted Plasmid pFBAIN-YLDGAT2  20 (8532bp) Plasmid pKS123  21 (7049 bp) cal a24-4  22 (1098 bp) Plasmid pKR53B 25 (8138 bp) Plasmid pKR72  26 (7085 bp) Plasmid pKR85  27 (7085 bp)Plasmid pPCR85  30 (4827 bp) Plasmid pKR91  31 (15114 bp) Plasmid pKR92 32 (13268 bp) Plasmid pKR92 YL DGAT2,  33 pKR92 with the (19604 bp) YLDGAT2 gene inserted Plasmid pKR92 YL DGAT1  34 YL DGAT2 (20082 bp)Soybean glycinin 1 (GY1) gene  35 (Genbank X15121) (3527 bp) Soybean GY1promoter  36 (690 bp) Plasmid pZBL114  39 (6660 bp) Soybean GM GY1  40(glycinin 1) gene (1437 bp) Synthetic BHL8 (barley high  41 lysine) gene(204 bp) GY1-BHL8 fusion product  42 (1701 bp) Plasmid pZBL133  43 (6493bp) Plasmid pKS238  44 (6472 bp) Plasmid pKS240  45 (6259 bp) PlasmidpKS120  46 (5267 bp) Plasmid pKS242  47 (8643 bp) Plasmid pKS349  48(8720 bp) Plasmid pKS121/BS  49 (5280 bp) Plasmid pDs-Red in pKS121/BS 50 (5968 bp) Plasmid pKS332  51 (10058 bp) Plasmid pKS362comprisingwild-  52 type Yarrowia lipolytica (11611 bp) DGAT2 driven bya beta- conglycinin promoter Soybean promoter GM P34  53 (1422 bp)Plasmid pZBL115  56 (7466 bp) Plasmid pJS89  57 (7841 bp) Morteriellaalpina delta-6  58 desaturase gene coding sequence (1390 bp) PlasmidpJS93  59 (9223 bp) Plasmid pKS127  60 (7472 bp) Plasmid pKS343  61(7847 bp) Plasmid pKS352  62 (10866 bp) Plasmid pKS364  63 (12055 bp)Yarrowia lipolytica DGAT1  64 65 gene codon optimized for soybean (1581bp) (526aa) Yarrowia lipolytica DGAT2  66 67 gene codon optimized forsoybean (1545 bp) (514aa) Plasmid pKR1234  68 (8638 bp) PlasmidppPSgly32  71 (3673 bp) Plasmid pKR264  72 (4171 bp) Plasmid pKR1212  73(6130 bp) Plasmid pKR1235  74 (5764 bp) Plasmid pKR1236 comprising  75both Yarrowia lipolytica (11693 bp) DGAT1 and DGAT2 Plasmid pKR1254comprising  78 wild-type Yarrowia lipolytica (5079 bp) DGAT2 PlasmidpKR1254_Y326F,  81 pKR1254 comprising mutant (5079 bp) Y326F Yarrowialipolytica DGAT2 Yarrowia lipolytica DGAT2  82 83 comprising codon 326mutated (1545 bp) (514aa) from Tyr to Phe Plasmid pKR1254_Y326L,  86pKR1254 comprising mutant (5079 bp) Y326L Yarrowia lipolytica DGAT2Yarrowia lipolytica DGAT2  87 88 comprising codon 326 mutated (1545 bp)(514aa) from Tyr to Leu Plasmid pKR1254_R327K, 91 pKR1254 comprisingmutant (5079 bp) R327K Yarrowia lipolytica DGAT2 Yarrowia lipolyticaDGAT2  92 93 comprising codon 327 mutated (1545 bp) (514aa) from Arg toLys Plasmid pY191 yeast expression  94 vector comprising wild-type (9074bp) Yarrowia lipolytica DGAT2 Plasmid pY192 yeast expression  95 vectorcomprising mutant Y326F (9074 bp) Yarrowia lipolytica DGAT2 PlasmidpY193 yeast expression  96 vector comprising mutant Y326L (9074 bp)Yarrowia lipolytica DGAT2 Plasmid pY194 yeast expression  97 vectorcomprising mutant R327K (9074 bp) Yarrowia lipolytica DGAT2 PlasmidpKR1256 soybean 98 expression vector comprising (8641 bp) wild-typeYarrowia lipolytica DGAT2 Plasmid pKR1277 soybean 99 expression vectorcomprising (8641 bp) mutant Y326F Yarrowia lipolytica DGAT2 PlasmidpKR1278 soybean 100  expression vector comprising (8641 bp) mutant Y326LYarrowia lipolytica DGAT2 Plasmid pKS392 comprising 101 Yarrowialipolytica DGAT1 (11647 bp) codon optimized for soybean driven byb-conglycinin promoter Plasmid pKS393 comprising 102 Yarrowia lipolyticaDGAT2 (11611 bp) codon optimized for soybean driven by b-con promoterPlasmid pKS391 comprising 103 wild-type Yarrowia lipolytica (11649 bp)DGAT1 driven by b-con promoter Plasmid pFBAIn-YLPAT1 104 (7674 bp)Plasmid pKR1239 105 (4391 bp) Plasmid pKR457 106 (5252 bp) PlasmidpKR1242 107 (5952 bp) Plasmid pFBAIn-YLPAAT2 108 (7836 bp) PlasmidpKR1240 109 (4553 bp) Plasmid pKR1243 110 (6114 bp) PlasmidpFBAIn-YLPAT3 111 (8229 bp) Plasmid pKR1241 112 (4946 bp) PlasmidpKR1244 113 (6507 bp) Plasmid pY27 114 (8928 bp) Plasmid pKR1246 115(7210 bp) Plasmid pYAT-ACBP yeast 116 expression vector comprising (7069bp) Yarrowia lipolytica ACBP (acyl-CoA binding protein) Plasmid pKR1245117 (3965 bp) Plasmid pKR1250 soybean 118 expression vector comprising(5526 bp) Yarrowia lipolytica ACBP (acyl-CoA binding protein) PlasmidpFBAIn-YCPT1 119 (8172 bp) Plasmid pKR1247 120 (4889 bp) Plasmid pKR1251121 (6450 bp) Yarrowia lipolytica GPAT 122 123  (glycerolphosphateacyltransferase (2184 bp) (727aa) homolog) Plasmid PCRblunt-YLGPAT 126(5715 bp) Plasmid pKR1257 127 (7447 bp) Yarrowia lipolytica PAP1 128129  (phosphatidic acid phosphatase (837 bp) (278aa) homolog 1) PlasmidpHD33 132 (3875 bp) Plasmid pKR1347 133 (6100 bp) Yarrowia lipolyticaPAP3 134 135  (phosphatidic acid phosphatase (1602 bp) (533aa) homolog3) Plasmid pHD34 138 (4640 bp) Plasmid pKR1348-2 139 (6865 bp) Yarrowialipolytica PAP2 140 141  (phosphatidic acid phosphatase (2172 bp)(723aa) homolog 2) Plasmid pHD35 144 (5210 bp) Plasmid pKR1349 145 (7435bp) Yarrowia lipolytica GPD (glycerol 146 147  phosphate dehydrogenasehomolog) (1197 bp) (398aa) Plasmid pHD36 152 (4729 bp) Plasmid pKR1273153 (5249 bp) Plasmid pKR1369 154 (6457 bp) Soybean (Glycine max)LPAAT-like 155 156  (lysophosphatidic acid (1122 bp) (373aa)acyltransferase-like homolog) Plasmid pKR561 soy LPAAT-like 159 (8654bp) Catalpa speciosa LPAAT-like 160 161  (lysophosphatidic acid (1116bp) (371aa) acyltransferase-like homolog) Plasmid pKR561 catalpa 164LPAAT-like (8653 bp) Plasmid pKR268 165 (4906 bp) Plasmid pKR145 166(7096 bp) Plasmid pKR561 167 (7497 bp) Plasmid pKS387 168 (13308 bp)Plasmid pKS178 169 (6498 bp) Plasmid pKR1358 170 (6063 bp) PlasmidpKR1364 171 (8936 bp) Plasmid pYAT MOD1 180 (7043 bp) Yarrowialipolytica ACBP 181 182  (acyl-CoA binding protein) (261 bp) (86aa)Yarrowia lipolytica LPAAT1 185 186  (lysophosphatidic acid (672 bp)(223aa) acyltransferase-1) Yarrowia lipolytica LPAAT1 187 188 (lysophosphatidic acid (849 bp) (282aa) acyltransferase-2) Yarrowialipolytica LPAAT1 189 190  (lysophosphatidic acid (1230 bp) (409aa)acyltransferase-3) Yarrowia lipolytica PDAT 191 192  (phospholipid:diacylglyceride (1944 bp) (648aa) acyltransferase) Yarrowia lipolyticaCPT (choline 193 194  phosphotransferase) (1185 bp) (394aa) PlasmidpKR278 195 (5303 bp) Plasmid pKR1274 196 (8358 bp) Soybean (Glycine max)197 thioesterase 2 (TE2) (1251 bp) Plasmid pTC4 198 (9592 bp) PlasmidpKR1258 203 (4738 bp) Soybean (Glycine max) fatty acid 204 desaturase2-1 (Fad2-1)) (1164 bp) Plasmid pBS43 205 (10303 bp) PlasmidPCRblunt-Fad2-1 210 (4584 bp) Plasmid pKR1259 211 (5797 bp) PlasmidpKR1261 212 (7590 bp) Plasmid pKR123R 213 (4993 bp) Plasmid pKR1266 214(9036 bp) Plasmid pKR1267 215 (11615 bp) Plasmid pKR457 216 (5252 bp)Plasmid pKR1264 217 (9295 bp) Plasmid pKR1277 218 (2577 bp) PlasmidpKR1269 219 (9219 bp) plasmid SH58 220 (8819) plasmid pJMS33 224 (9396)plasmid PHP25069 (SH74) 225 (11440) plasmid pJMS38 229 (11111) plasmidPHP21155 230  (3231) plasmid PHP29252 (pJMS41) 235 (11879) plasmidPHP19031A 236  (3984)

SEQ ID NOs:7-8 correspond to PCR primers oYLDGAT2-1 (SEQ IOD NO:7) andoYLDGAT2-2 (SEQ IOD NO:8), used to amplify the Yarrowia lipolyticadiacylglycerol acyltransferase 2 (YL DGAT2) gene from a yeast lysate(for details see Example 1.)

SEQ ID NOs:12-15 correspond to oligonucleotide primers used to amplifythe coding regions of YL DGAT1 (YDGAT1-F and YDGAT1-R; SEQ ID NOs:12-13,respectively) and YL DGAT2 (YDGAT2-F and YDGAT2-R, SEQ ID NOs:14-15,respectively) from Yarrowia lipolytica genomic DNA.

SEQ ID NOs:23 (oCal-15) and SEQ ID NO:24 (oCal-6) correspond tooligonucleotide primers used to amplify DNA fragment cal a24-4 (SEQ IDNO:22) from template plasmid CaIFad2-2 described in PCT Publication No.WO 02/008269.

SEQ ID NO:28 (oKR85-1) and SEQ ID NO:29 (oKR85-2) correspond to primersused to amplify the beta-conglycinin promoter-(NotI cloningsite)-phaseolin 3′ terminator region from plasmid pKR85 (SEQ ID NO:27.)

SEQ ID NOs:37(oGy1-1) and SEQ ID NO:38 (oGy1-2) correspond to primersused to amplify the soybean glycinin 1 promoter (SEQ ID NO:36) andincorporating BamHI and NcoI sites on the 5′ and 3′-ends, respectively.

SEQ ID NO:54 (oP34-1) and SEQ ID NO:55 (oP34-2) correspond to primersused to amplify the soybean P34 promoter (SEQ ID NO:53) andincorporating BamHI and NotI sites into the 5′ and 3′-ends,respectively.

SEQ ID NO:69 (oSGly-2) and SEQ ID NO:70 (oSGly-3) correspond to primersused to amplify the glycinin GY1 promoter.

SEQ ID NOs:76(oYDG2-1) and SEQ ID NO:77 (oYDG2-2) correspond to primersused to amplify Yarrowia DGAT2 (SEQ ID NO:10) which was thenincorporated into pKR1254 (SEQ ID NO:78).

SEQ ID NO:79 (YID2_Y326F-5) and SEQ ID NO:80 (YID2_Y326F-3) correspondto primers used to mutate the amino acid at position 326 of YarrowiaDGAT2 (SEQ ID NO:10) from tyrosine to phenylalanine.

SEQ ID NO:84 (YID2_Y326L-5) and SEQ ID NO:85 (YID2_Y326L-3) correspondto primers used to mutate the amino acid at position 326 of YarrowiaDGAT2 (SEQ ID NO:10) from tyrosine to leucine.

SEQ ID NO:89 (YID2_R327K-5) and SEQ ID NO:90 (YID2_R327K-3) correspondto primers used to mutate the amino acid at position 327 of YarrowiaDGAT2 (SEQ ID NO:10) from arginine to lysine.

SEQ ID NOs:124 (YIGPAT-5) and SEQ ID NO:125 (YIGPAT-3) correspond toprimers used to amplify the Yarrowia glycerolphosphate acyltransferasehomolog (YL GPAT) which was then incorporated into pKR1257 (SEQ IDNO:127).

SEQ ID NOs:130 (YIPAP1-5) and SEQ ID NO:131 (YIPAP1-3) correspond toprimers used to amplify the Yarrowia phosphatidic acid phosphatasehomolog 1 (YL PAP1) which was then incorporated into pKR1347 (SEQ IDNO:133).

SEQ ID NOs:136 (YIPAP3-5) and SEQ ID NO:137 (YIPAP3-3) correspond toprimers used to amplify the Yarrowia phosphatidic acid phosphatasehomolog 3 (YL PAP3) which was then incorporated into pKR1348_2 (SEQ IDNO:139).

SEQ ID NOs:142 (YIPAP2-5) and SEQ ID NO:143 (YIPAP2-3) correspond toprimers used to amplify the Yarrowia phosphatidic acid phosphatasehomolog 2 (YL PAP2) which was then incorporated into pKR1349 (SEQ IDNO:145).

SEQ ID NOs:148 (YIGPD-5) and SEQ ID NO:149 (YIGPD-3-2) and SEQ IDNOs:150 (YIGPD-intron-5-2) and SEQ ID NO:151 (YIGPD-3) correspond toprimers used to amplify the Yarrowia glycerol phosphate dehydrogenasehomolog (YL GPD) which was then incorporated into pKR1273 (SEQ IDNO:153).

SEQ ID NOs:157 (soy LPAAT-like fwd) and SEQ ID NO:158 (soy LPAAT-likerev) correspond to primers used to amplify the soybean lysophosphatidicacid acyltransferase-like homolog (LPAAT-like; SEQ ID NO:156) which wasthen incorporated into pKR561-SOY LPAAT-like (SEQ ID NO:159).

SEQ ID NOs:162 (catalpa LPAAT-like fwd) and SEQ ID NO:163 (catalpaLPAAT-like rev) correspond to primers used to amplify the catalpalysophosphatidic acid acyltransferase-like homolog (LPAAT-like; SEQ IDNO:161) which was then incorporated into pKR561-CATALPA LPAAT-like (SEQID NO:164).

SEQ ID NOs:172 (YL LPAAT1 fwd) and SEQ ID NO:173 (YL LPAAT1 rev)correspond to primers used to amplify the Yarrowia lysophosphatidic acidacyltransferase-1 homolog (YLPAT1) which was then incorporated intopFBAIn-YLPAT1 (SEQ ID NO:104).

SEQ ID NOs:174 (YL LPAAT2 fwd) and SEQ ID NO:175 (YL LPAAT2 rev)correspond to primers used to amplify the Yarrowia lysophosphatidic acidacyltransferase-2 homolog (YLPAT2) which was then incorporated intopFBAIn-YLPAAT2 (SEQ ID NO:108).

SEQ ID NOs:176 (YL LPAAT3 fwd) and SEQ ID NO:177 (YL LPAAT3 rev)correspond to primers used to amplify the Yarrowia lysophosphatidic acidacyltransferase-3 homolog (YLPAT3) which was then incorporated intopFBAIn-YLPAT3 (SEQ ID NO:111).

SEQ ID NOs:178 (YL ACBP fwd) and SEQ ID NO:179 (YL ACBP rev) correspondto primers used to amplify the Yarrowia acyl-CoA binding protein homolog(YL ACBP) which was then incorporated into pYAT-ACBP (SEQ ID NO:116).

SEQ ID NOs:183 (YL CPT fwd) and SEQ ID NO:184 (YL CPT rev) correspond toprimers used to amplify the Yarrowia phosphotransferase homolog (YL CPT)which was then incorporated into—YCPT1 (SEQ ID NO:119).

SEQ ID NOs:199 (GmTE2-1 5-1) and SEQ ID NO:200 (GmTE2-1 3-1) and SEQ IDNOs:201 (GmTE2-1 5-2) and SEQ ID NO:202 (GmTE2-1 3-2) correspond toprimers used to amplify the soybean (Glycine max) thioesterase 2 gene(TE2) which was then incorporated into pKR1258 (SEQ ID NO:203).

SEQ ID NO:206 (GmFad2-1 5-1) and SEQ ID NO:207 (GmFad2-1 3-1) and

SEQ ID NOs:208 (GmFad2-1 5-2) and SEQ ID NO:209 (GmFad2-1 3-2)correspond to primers used to amplify the soybean (Glycine max) fattyacid desaturase 2-1 gene (Fad2-1) which was then incorporated intopKR1259 (SEQ ID NO:211).

SEQ ID NO:221 (G1 HPFW04) and SEQ ID NO:222 (G3HPRV02) are primers usedto PCR amplify fragment H-Not1-Sal1-GAS1sGAS2GAS3-AvrII-B-H (SEQ IDNO:223, 1621 bp) which was digested and cloned into plasmid pJMS33 (SEQID NO:224).

SEQ ID NO:226 (GMPGMFW02) and SEQ ID NO:227 (GMPGMRV02) are primers usedto PCR amplify fragment PGMPCRAA (SEQ ID NO:228, 596 bp) which wasdigested and cloned into plasmid pJMS38 (SEQ ID NO:229).

SEQ ID NO:231 (PHP21155FW01) and SEQ ID NO:232 (PHP21155RV01) areprimers used to PCR amplify fragment STLS1(BsiWI-SbfI) (SEQ ID NO:233,220 bp) which was digested along with fragment PGMPCRAS (SEQ ID NO:234,592 bp) and cloned into plasmid pJMS41 (SEQ ID NO:235).

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

Acyl-CoA:sterol-acyltransferase” is abbreviated ARE2.

“Phospholipid:diacylglycerol acyltransferase” is abbreviated PDAT.

“Diacylglycerol acyltransferase” is abbreviated DAG AT or DGAT.

“Diacylglycerol” is abbreviated DAG.

“Triacylglycerols” are abbreviated TAGs.

“Co-enzyme A” is abbreviated CoA.

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂.

The structure of a fatty acid is represented by a simple notation systemof “X:Y”, where X is the total number of carbon (C) atoms in theparticular fatty acid and Y is the number of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9th and 10th carbon atom as for palmitoleic acid (16:1) andoleic acid (18:1)), while “polyunsaturated fatty acids” (or “PUFAs”)have at least two double bonds along the carbon backbone (e.g., betweenthe 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“Microbial oils” or “single cell oils” are those oils naturally producedby microorganisms (e.g., algae, oleaginous yeasts and filamentous fungi)during their lifespan. The term “oil” refers to a lipid substance thatis liquid at 25° C. and usually polyunsaturated. In contrast, the term“fat” refers to a lipid substance that is solid at 25° C. and usuallysaturated.

“Lipid bodies” refer to lipid droplets that usually are bounded byspecific proteins and a monolayer of phospholipid. These organelles aresites where most organisms transport/store neutral lipids. Lipid bodiesare thought to arise from microdomains of the endoplasmic reticulum thatcontain TAG-biosynthesis enzymes; and, their synthesis and size appearto be controlled by specific protein components.

“Neutral lipids” refer to those lipids commonly found in cells in lipidbodies as storage fats and oils and are so called because at cellularpH, the lipids bear no charged groups. Generally, they are completelynon-polar with no affinity for water. Neutral lipids generally refer tomono-, di-, and/or triesters of glycerol with fatty acids, also calledmonoacylglycerol, diacylglycerol or TAG, respectively (or collectively,acylglycerols). A hydolysis reaction must occur to release free fattyacids from acylglycerols.

The terms “triacylglycerol”, “oil” and “TAGs” refer to neutral lipidscomposed of three fatty acyl residues esterified to a glycerol molecule(and such terms will be used interchangeably throughout the presentdisclosure herein). Such oils can contain long chain PUFAs, as well asshorter saturated and unsaturated fatty acids and longer chain saturatedfatty acids. Thus, “oil biosynthesis” generically refers to thesynthesis of TAGs in the cell.

The term “DAG AT” or “DGAT” refers to a diacylglycerol acyltransferase(also known as an acyl-CoA-diacylglycerol acyltransferase or adiacylglycerol O-acyltransferase) (EC 2.3.1.20). This enzyme isresponsible for the conversion of acyl-CoA and 1,2-diacylglycerol to TAGand CoA (thereby involved in the terminal step of TAG biosynthesis). Twofamilies of DAG AT enzymes exist: DGAT1 and DGAT2. The former familyshares homology with the acyl-CoA:cholesterol acyltransferase (ACAT)gene family, while the latter family is unrelated (Lardizabal et al., J.Biol. Chem. 276(42):38862-28869 (2001)).

The term “PDAT” refers to a phospholipid:diacylglycerol acyltransferaseenzyme (EC 2.3.1.158). This enzyme is responsible for the transfer of anacyl group from the sn-2 position of a phospholipid to the sn-3 positionof 1,2-diacylglycerol, thus resulting in lysophospholipid and TAG(thereby involved in the terminal step of TAG biosynthesis). This enzymediffers from DGAT (EC 2.3.1.20) by synthesizing TAG via anacyl-CoA-independent mechanism.

The term “ARE2” refers to an acyl-CoA:sterol-acyltransferase enzyme (EC2.3.1.26; also known as a sterol-ester synthase 2 enzyme), catalyzingthe following reaction: acyl-CoA+cholesterol=CoA+cholesterol ester.

The term “Kennedy pathway enzyme genes” are defined as genes encodingenzymes that are involved in providing the immediate precursors formembrane lipid or storage lipid biosynthesis at the endoplasmicreticulum. Kennedy pathway enzymes also include enzymes that catalyzetransfer of acyl groups between intermediates of membrane lipid or seedstorage lipid biosynthesis at the endoplasmic reticulum (ER). Kennedypathway enzyme can be soluble, cytosolic enzymes. They can associatedwith the ER membrane system or they can be integral membrane proteins ofthe ER membrane system. A “Kennedy Pathway gene” is further defined asany gene directly involved biosynthesis or degradation oftriacylglycerol (TAG) or TAG intermediates. Some examples of genesinclude glycerol-phosphate dehydrogenase (GPD), glycerol-phosphateacyltransferase (G PAT), glycerol acyltransferase, lyso-phospholipidacyltransferase (LPAT), lyso-phosphatidic acid acyltransferase (LPAAT),lyso-phosphatidylcholine acyltransferase (LPCAT), monoacylglycerideacyltransferase, phosphatidic acid phosphatase (PAP), lyso-phospholipidphospholipase, lyso-phosphatidic acid phospholipase,lyso-phosphatidylcholine phospholipase, phospholipase A1 (PLA1),phospholipase A2 (PLA2), phospholipase B (PLB), phospholipase C (PLC),phospholipase D (PLD), choline phosphotransferase (CPT), plastidicphosphoglucomutase (PGM), phospholipid:diacylglyceride acyltransferase(PDAT), lyso-phospholipid:diglyceride acyltransferase (LPDAT),triacylglyceride lipase, diacylglyceride lipase, monoacylglyceridelipase, and acylCoA binding protein (ACBP).

As used herein, “nucleic acid” means a polynucleotide and includessingle or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Thus, the terms “polynucleotide”, “nucleic acidsequence”, “nucleotide sequence” or “nucleic acid fragment” are usedinterchangeably and is a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. Nucleotides (usually found in their 5′-monophosphateform) are referred to by their single letter designation as follows: “A”for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deosycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridlate, “T” for deosythymidylate, “R” for purines (A or G), “Y”for pyrimidiens (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The terms “subfragment that is functionally equivalent” and“functionally equivalent subfragment” are used interchangeably herein.These terms refer to a portion or subsequence of an isolated nucleicacid fragment in which the ability to alter gene expression or produce acertain phenotype is retained whether or not the fragment or subfragmentencodes an active enzyme. For example, the fragment or subfragment canbe used in the design of chimeric genes to produce the desired phenotypein a transformed plant. Chimeric genes can be designed for use insuppression by linking a nucleic acid fragment or subfragment thereof,whether or not it encodes an active enzyme, in the sense or antisenseorientation relative to a plant promoter sequence.

The term “conserved domain” or “motif” means a set of amino acidsconserved at specific positions along an aligned sequence ofevolutionarily related proteins. While amino acids at other positionscan vary between homologous proteins, amino acids that are highlyconserved at specific positions indicate amino acids that are essentialin the structure, the stability, or the activity of a protein.

Because they are identified by their high degree of conservation inaligned sequences of a family of protein homologues, they can be used asidentifiers, or “signatures”, to determine if a protein with a newlydetermined sequence belongs to a previously identified protein family.

The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases do not affect the ability of the nucleic acid fragmentto mediate gene expression or produce a certain phenotype. These termsalso refer to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotides thatdo not substantially alter the functional properties of the resultingnucleic acid fragment relative to the initial, unmodified fragment. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize (under moderately stringent conditions, e.g.,0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplified herein, or toany portion of the nucleotide sequences disclosed herein and which arefunctionally equivalent to any of the nucleic acid sequences disclosedherein. Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 80% sequence identity, or 90% sequence identity, upto and including 100% sequence identity (i.e., fully complementary) witheach other.

The term “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a probe will selectivelyhybridize to its target sequence. Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, optionally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth et al., Anal. Biochem. 138:267-284 (1984): Tm=810.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)—500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Hybridization and/or washconditions can be applied for at least 10, 30, 60, 90, 120, or 240minutes.

“Sequence identity” or “identity” in the context of nucleic acid orpolypeptide sequences refers to the nucleic acid bases or amino acidresidues in two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window.

Thus, “percentage of sequence identity” refers to the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The percentage iscalculated by determining the number of positions at which the identicalnucleic acid base or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the results by 100 to yield the percentage of sequenceidentity. Useful examples of percent sequence identities include, butare not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%,or any integer percentage from 50% to 100%. These identities can bedetermined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations maybe determined using a variety of comparison methods designed to detecthomologous sequences including, but not limited to, the MegAlign™program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Within the context of this application it will beunderstood that where sequence analysis software is used for analysis,that the results of the analysis will be based on the “default values”of the program referenced, unless otherwise specified. As used herein“default values” will mean any set of values or parameters thatoriginally load with the software when first initialized.

The “Clustal V method of alignment” corresponds to the alignment methodlabeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153(1989); Higgins, D. G. et al. (1992) Comput. Appl. Biosci. 8:189-191)and found in the MegAlign™ program of the LASERGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). For multiple alignments,the default values correspond to GAP PENALTY=10 and GAP LENGTHPENALTY=10. Default parameters for pairwise alignments and calculationof percent identity of protein sequences using the Clustal method areKTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleicacids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 andDIAGONALS SAVED=4. After alignment of the sequences using the Clustal Vprogram, it is possible to obtain a “percent identity” by viewing the“sequence distances” table in the same program.

“BLASTN method of alignment” is an algorithm provided by the NationalCenter for Biotechnology Information (NCBI) to compare nucleotidesequences using default parameters.

It is well understood by one skilled in the art that many levels ofsequence identity are useful in identifying polypeptides, from otherspecies, wherein such polypeptides have the same or similar function oractivity. Useful examples of percent identities include, but are notlimited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or anyinteger percentage from 50% to 100%. Indeed, any integer amino acididentity from 50% to 100% may be useful in describing the presentinvention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Also, ofinterest is any full-length or partial complement of this isolatednucleotide fragment.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes. A “transgene” is a gene that hasbeen introduced into the genome by a transformation procedure.

The term “genome” as it applies to a plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondrial, plastid) of the cell.

A “codon-optimized gene” is a gene having its frequency of codon usagedesigned to mimic the frequency of preferred codon usage of the hostcell.

An “allele” is one of several alternative forms of a gene occupying agiven locus on a chromosome. When all the alleles present at a givenlocus on a chromosome are the same that plant is homozygous at thatlocus. If the alleles present at a given locus on a chromosome differthat plant is heterozygous at that locus.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to: promoters, translation leader sequences, introns,polyadenylation recognition sequences, RNA processing sites, effectorbinding sites and stem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence that can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental conditions. It is furtherrecognized that since in most cases the exact boundaries of regulatorysequences have not been completely defined, DNA fragments of somevariation may have identical promoter activity. Promoters that cause agene to be expressed in most cell types at most times are commonlyreferred to as “constitutive promoters”. New promoters of various typesuseful in plant cells are constantly being discovered; numerous examplesmay be found in the compilation by Okamuro, J. K., and Goldberg, R. B.Biochemistry of Plants 15:1-82 (1989).

“Translation leader sequence” refers to a polynucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., Mol.Biotechnol. 3:225-236 (1995)).

“3′ non-coding sequences”, “transcription terminator” or “terminationsequences” refer to DNA sequences located downstream of a codingsequence and include polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al. Plant Cell1:671-680 (1989).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript. A RNA transcript is referred toas the mature RNA when it is a RNA sequence derived frompost-transcriptional processing of the primary transcript. “MessengerRNA” or “mRNA” refers to the RNA that is without introns and that can betranslated into protein by the cell. “cDNA” refers to a DNA that iscomplementary to, and synthesized from, a mRNA template using the enzymereverse transcriptase. The cDNA can be single-stranded or converted intodouble-stranded form using the Klenow fragment of DNA polymerase I.“Sense” RNA refers to RNA transcript that includes the mRNA and can betranslated into protein within a cell or in vitro. “Antisense RNA”refers to an RNA transcript that is complementary to all or part of atarget primary transcript or mRNA, and that blocks the expression of atarget gene (U.S. Pat. No. 5,107,065). The complementarity of anantisense RNA may be with any part of the specific gene transcript,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to antisense RNA, ribozymeRNA, or other RNA that may not be translated but yet has an effect oncellular processes. The terms “complement” and “reverse complement” areused interchangeably herein with respect to mRNA transcripts, and aremeant to define the antisense RNA of the message.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis regulated by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of regulating the expressionof that coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in a sense or antisenseorientation. In another example, the complementary RNA regions of theinvention can be operably linked, either directly or indirectly, 5′ tothe target mRNA, or 3′ to the target mRNA, or within the target mRNA, ora first complementary region is 5′ and its complement is 3′ to thetarget mRNA.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989).Transformation methods are well known to those skilled in the art andare described infra.

“PCR” or “polymerase chain reaction” is a technique for the synthesis oflarge quantities of specific DNA segments and consists of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double-stranded DNA is heat denatured, the two primerscomplementary to the 3′ boundaries of the target segment are annealed atlow temperature and then extended at an intermediate temperature. Oneset of these three consecutive steps is referred to as a “cycle”.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitates transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host (i.e., to a discretenucleic acid fragment into which a nucleic acid sequence or fragment canbe moved.)

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, a chimericconstruct may comprise regulatory sequences and coding sequences thatare derived from different sources, or regulatory sequences and codingsequences derived from the same source, but arranged in a mannerdifferent than that found in nature. Such a construct may be used byitself or may be used in conjunction with a vector. If a vector is used,then the choice of vector is dependent upon the method that will be usedto transform host cells as is well known to those skilled in the art.For example, a plasmid vector can be used. The skilled artisan is wellaware of the genetic elements that must be present on the vector inorder to successfully transform, select and propagate host cellscomprising any of the isolated nucleic acid fragments of the invention.The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al.,Mol. Gen. Genetics 218:78-86 (1989)), and thus that multiple events mustbe screened in order to obtain lines displaying the desired expressionlevel and pattern. Such screening may be accomplished by Southernanalysis of DNA, Northern analysis of mRNA expression, immunoblottinganalysis of protein expression, or phenotypic analysis, among others.

The term “expression”, as used herein, refers to the production of afunctional end-product (e.g., a mRNA or a protein [either precursor ormature]).

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Mature” protein refers to a post-translationally processed polypeptide(i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed). “Precursor” protein refers tothe primary product of translation of mRNA (i.e., with pre- andpropeptides still present). Pre- and propeptides may be but are notlimited to intracellular localization signals.

“Stable transformation” refers to the transfer of a nucleic acidfragment into a genome of a host organism, including both nuclear andorganellar genomes, resulting in genetically stable inheritance. Incontrast, “transient transformation” refers to the transfer of a nucleicacid fragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without integration or stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms.

As used herein, “transgenic” refers to a plant or a cell which compriseswithin its genome a heterologous polynucleotide. Preferably, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of an expression construct. Transgenic is used herein to includeany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenics initially so altered as well as thosecreated by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Co-suppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of identical or substantiallysimilar foreign or endogenous genes (U.S. Pat. No. 5,231,020).Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence(Vaucheret et al., Plant J. 16:651-659 (1998); Gura, Nature 404:804-808(2000)). The overall efficiency of this phenomenon is low, and theextent of the RNA reduction is widely variable. More recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication No. WO 99/53050, published Oct. 21, 1999; PCT PublicationNo. WO 02/00904, published Jan. 3, 2002). This increases the frequencyof co-suppression in the recovered transgenic plants. Another variationdescribes the use of plant viral sequences to direct the suppression, or“silencing”, of proximal mRNA encoding sequences (PCT Publication No. WO98/36083, published Aug. 20, 1998). Both of these co-suppressingphenomena have not been elucidated mechanistically, although geneticevidence has begun to unravel this complex situation (Elmayan et al.,Plant Cell 10:1747-1757 (1998)).

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2nd Ed., Plenum, 1980). A class of plants identified asoleaginous are commonly referred to as “oilseed” plants. Examples ofoilseed plants include, but are not limited to: soybean (Glycine andSoja sp.), flax (Linum sp.), rapeseed (Brassica sp.), maize, cotton,safflower (Carthamus sp.) and sunflower (Helianthus sp.).

Within oleaginous microorganisms the cellular oil or TAG contentgenerally follows a sigmoid curve, wherein the concentration of lipidincreases until it reaches a maximum at the late logarithmic or earlystationary growth phase and then gradually decreases during the latestationary and death phases (Yongmanitchai and Ward, Appl. Environ.Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that make oil. It is not uncommon for oleaginous microorganismsto accumulate in excess of about 25% of their dry cell weight as oil.Examples of oleaginous yeast include, but are no means limited to, thefollowing genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces.

The term “plant” refers to whole plants, plant organs, plant tissues,seeds, plant cells, seeds and progeny of the same. Plant cells include,without limitation, cells from seeds, suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen and microspores.

“Progeny” comprises any subsequent generation of a plant.“Non-transgenic, null segregant soybean seed” refers to a near isogenicplant or seed that lacks the transgene, and/or a parental plant used inthe transformation process to obtain the transgenic event. Nullsegregants can be plants or seed that do not contain the transgenictrait due to normal genetic segregation during propagation of theheterozygous transgenic plants.

A “kernel” is the corn caryopsis, consisting of a mature embryo andendosperm which are products of double fertilization. The term “corn” or“maize” represents any variety, cultivar, or population of Zea mays L.

“Grain” comprises mature corn kernels produced by commercial growers foron farm use or for sale to customers in both cases for purposes otherthan growing or reproducing the species. The “seed” is the mature cornkernel produced for the purpose of propagating the species and for saleto commercial growers. As used herein the terms seeds, kernels, andgrains can be used interchangeably. The “embryo” or also termed “germ”is a young sporophytic plant, before the start of a period of rapidgrowth (seed germination). The embryo (germ) of corn contains the vastmajority of the oil found in the kernel. The structure of embryo incereal grain includes the embryonic axis and the scutellum. The“scutellum” is the single cotyledon of a cereal grain embryo,specialized for absorption of the endosperm. The “aleurone” is aproteinaceous material, usually in the form of small granules, occurringin the outermost cell layer of the endosperm of corn and other grains.

The present invention concerns a transgenic soybean seed havingincreased total fatty acid content of at least 20% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed. It is understood that any measurable increase in the total fattyacid content of a transgenic versus a non-transgenic, null segregantwould be useful. Such increases in the total fatty acid content wouldinclude, but are not limited to, at least 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or40%.

A transgenic oilseed of the invention can comprise a recombinantconstruct having at least one DGAT sequence. This DGAT sequence can beselected from the group consisting of DGAT1, DGAT2 and DGAT1 incombination with DGAT2. Furthermore, at least one DGAT sequence can befrom Yarrowia. Examples of suitable DGAT sequences that can be used topractice the invention are discussed in the Examples below. There can bementioned SEQ ID NOs:1, 9, 64, 66, 82, 87, and 92 in the presentinvention. Those skilled in the art will appreciate that the instantinvention includes, but is not limited to, the DGAT sequences disclosedherein.

Such a recombinant construct promoter would comprise differentcomponents such as a promoter which is a DNA sequence that directscellular machinery of a plant to produce RNA from the contiguous codingsequence downstream (3′) of the promoter. The promoter region influencesthe rate, developmental stage, and cell type in which the RNA transcriptof the gene is made. The RNA transcript is processed to produce mRNAwhich serves as a template for translation of the RNA sequence into theamino acid sequence of the encoded polypeptide. The 5′ non-translatedleader sequence is a region of the mRNA upstream of the protein codingregion that may play a role in initiation and translation of the mRNA.The 3′ transcription termination/polyadenylation signal is anon-translated region downstream of the protein coding region thatfunctions in the plant cell to cause termination of the RNA transcriptand the addition of polyadenylate nucleotides to the 3′ end of the RNA.

The origin of the promoter chosen to drive expression of the DGAT codingsequence is not important as long as it has sufficient transcriptionalactivity to accomplish the invention by expressing translatable mRNA forthe desired nucleic acid fragments in the desired host tissue at theright time. Either heterologous or non-heterologous (i.e., endogenous)promoters can be used to practice the invention. For example, suitablepromoters include, but are not limited to: the alpha prime subunit ofbeta conglycinin promoter, the Kunitz trypsin inhibitor 3 promoter, theannexin promoter, the glycinin Gyl promoter, the beta subunit of betaconglycinin promoter, the P34/Gly Bd m 30K promoter, the albuminpromoter, the Leg A1 promoter and the Leg A2 promoter.

The annexin, or P34, promoter is described in PCT Publication No. WO2004/071178 (published Aug. 26, 2004). The level of activity of theannexin promoter is comparable to that of many known strong promoters,such as: (1) the CaMV 35S promoter (Atanassova et al., Plant Mol. Biol.37:275-285 (1998); Battraw and Hall, Plant Mol. Biol. 15:527-538 (1990);Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); Jefferson et al.,EMBO J. 6:3901-3907 (1987); Wilmink et al., Plant Mol. Biol. 28:949-955(1995)); (2) the Arabidopsis oleosin promoters (Plant et al., Plant Mol.Biol. 25:193-205 (1994); Li, Texas A & M University Ph.D. dissertation,pp. 107-128 (1997)); (3) the Arabidopsis ubiquitin extension proteinpromoters (Callis et al., J Biol. Chem. 265(21):12486-93 (1990)); (4) atomato ubiquitin gene promoter (Rollfinke et al., Gene. 211(2):267-76(1998)); (5) a soybean heat shock protein promoter (Schoffl et al., MolGen Genet. 217(2-3):246-53 (1989)); and, (6) a maize H3 histone genepromoter (Atanassova et al., Plant Mol Biol. 37(2):275-85 (1989)).

Another useful feature of the annexin promoter is its expression profilein developing seeds. The annexin promoter is most active in developingseeds at early stages (before 10 days after pollination) and is largelyquiescent in later stages. The expression profile of the annexinpromoter is different from that of many seed-specific promoters, e.g.,seed storage protein promoters, which often provide highest activity inlater stages of development (Chen et al., Dev. Genet. 10:112-122 (1989);Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996); Keddie et al.,Plant Mol. Biol. 24:327-340 (1994); Plant et al., (supra); Li, (supra)).The annexin promoter has a more conventional expression profile butremains distinct from other known seed specific promoters. Thus, theannexin promoter will be a very attractive candidate whenoverexpression, or suppression, of a gene in embryos is desired at anearly developing stage. For example, it may be desirable to overexpressa gene regulating early embryo development or a gene involved in themetabolism prior to seed maturation.

Following identification of an appropriate promoter suitable forexpression of a specific DGAT-coding sequence, the promoter is thenoperably linked in a sense orientation using conventional means wellknown to those skilled in the art.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2nd ed.; Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1989 (hereinafter“Sambrook et al., 1989”) or Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K., Eds.; InCurrent Protocols in Molecular Biology; John Wiley and Sons: New York,1990 (hereinafter “Ausubel et al., 1990”).

This invention concerns a method for increasing the total fatty acidcontent of a soybean seed comprising:

(a) transforming at least one soybean cell with a recombinant constructhaving at least one DGAT sequence;

(b) selecting the transformed soybean cell(s) of step (a) having anincreased total fatty acid content of at least 20% when compared to thetotal fatty acid content of a non-transgenic, null segregant soybeanseed.

In another aspect, this invention concerns combining, either on the sameconstruct, or a separate construct, at least on DGAT sequence with aconstruct causing the downregulation of plastidic phosphoglucomutase.The downregulation of plastidic phosphoglucomutase activity can occurby, but is not limited to, cosuppression, antisense, microRNA targeting,or translational inhibition.

Once the recombinant construct has been made, it may then be introducedinto a plant cell of choice by methods well known to those of ordinaryskill in the art (e.g., transfection, transformation andelectroporation). Oilseed plant cells are the preferred plant cells. Thetransformed plant cell is then cultured and regenerated under suitableconditions permitting selection of those transformed soybean cell(s)having an increased total fatty acid content of at least 20% whencompared to the total fatty acid content of a non-transgenic, nullsegregant soybean seed.

Such recombinant constructs may be introduced into one plant cell; or,alternatively, each construct may be introduced into separate plantcells.

Expression in a plant cell may be accomplished in a transient or stablefashion as is described above.

Also within the scope of this invention are seeds or plant partsobtained from such transformed plants.

Plant parts include differentiated and undifferentiated tissuesincluding, but not limited to the following: roots, stems, shoots,leaves, pollen, seeds, tumor tissue and various forms of cells andculture (e.g., single cells, protoplasts, embryos and callus tissue).The plant tissue may be in plant or in a plant organ, tissue or cellculture.

The term “plant organ” refers to plant tissue or a group of tissues thatconstitute a morphologically and functionally distinct part of a plant.The term “genome” refers to the following: (1) the entire complement ofgenetic material (genes and non-coding sequences) that is present ineach cell of an organism, or virus or organelle; and/or (2) a completeset of chromosomes inherited as a (haploid) unit from one parent.

Methods for transforming dicots (primarily by use of Agrobacteriumtumefaciens) and obtaining transgenic plants have been published, amongothers, for: cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al. Plant Cell Rep.15:653-657 (1996); McKently et al. Plant Cell Rep. 14:699-703 (1995));papaya (Ling, K. et al. Bio/technology 9:752-758 (1991)); and pea (Grantet al. Plant Cell Rep. 15:254-258 (1995)). For a review of othercommonly used methods of plant transformation see Newell, C. A. (Mol.Biotechnol. 16:53-65 (2000)). One of these methods of transformationuses Agrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F.Microbiol. Sci. 4:24-28 (1987)). Transformation of soybeans using directdelivery of DNA has been published using PEG fusion (PCT Publication No.WO 92/17598), electroporation (Chowrira, G. M. et al., Mol. Biotechnol.3:17-23 (1995); Christou, P. et al., Proc. Natl. Acad. Sci. U.S.A.84:3962-3966 (1987)), microinjection and particle bombardement (McCabe,D. E. et. al., Bio/Technology 6:923 (1988); Christou et al., PlantPhysiol. 87:671-674 (1988)).

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, In: Methodsfor Plant Molecular Biology, (Eds.), Academic: San Diego, Calif.(1988)). This regeneration and growth process typically includes thesteps of selection of transformed cells and culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for: the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.); thegeneration of recombinant DNA fragments and recombinant expressionconstructs; and, the screening and isolating of clones. See, forexample: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor: NY (1989); Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor: NY (1995); Birren et al., Genome Analysis:Detecting Genes, Vol. 1, Cold Spring Harbor: NY (1998); Birren et al.,Genome Analysis: Analyzing DNA, Vol. 2, Cold Spring Harbor: NY (1998);Plant Molecular Biology: A Laboratory Manual, eds. Clark, Springer: NY(1997).

Examples of oilseed plants include, but are not limited to: soybean,Brassica species, sunflower, maize, cotton, flax and safflower.

As was discussed above, any of the transgenic oilseeds discussed hereincan comprise a recombinant construct having at least one DGAT sequence.This DGAT sequence can be selected from the group consisting of DGAT1,DGAT2, or DGAT1 in combination with DGAT2. Furthermore, at least oneDGAT sequence is from Yarrowia.

Transformation of monocotyledons using electroporation, particlebombardment, and Agrobacterium have been reported. Transformation andplant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354, (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); Zea mays (Rhodes et al., Science 240:204(1988), Gordon-Kamm et al., Plant Cell 2:603-618 (1990), Fromm et al.,BiolTechnology 8:833 (1990), Koziel et al., BiolTechnology 11: 194,(1993), Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., BiolTechnology 10: 15 89 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., TheorAppl. Genet.205:34, (1986); Part et al., Plant Mol. Biol. 32:1135-1148, (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al. Plant Cell Rep.7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al., BiolTechnology 10:691 (1992)), and wheat(Vasil et al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al., EMBO J. 9:2517-2522 (1990)).

Transient expression systems may be used to functionally dissect geneconstructs (see generally, Maliga et al., Methods in Plant MolecularBiology, Cold Spring Harbor Press (1995)). It is understood that any ofthe nucleic acid molecules of the present invention can be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers etc.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant organisms and the screening and isolating ofclones, (see for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989); Maliga et al.,Methods in Plant Molecular Biology, Cold Spring Harbor Press (1995);Birren et al., Genome Analysis: Detecting Genes, 1, Cold Spring Harbor,N.Y. (1998); Birren et al., Genome Analysis: Analyzing DNA, 2, ColdSpring Harbor, N.Y. (1998); Plant Molecular Biology: A LaboratoryManual, eds. Clark, Springer, New York (1997)).

The transgenic soybean seeds of the invention can be processed to yieldsoy oil, soy products and/or soy by-products.

“Soy products” can include, but are not limited to, those items listedin Table 1A.

TABLE 1A Soy Protein Products Derived from Soybean Seeds^(a) WholeSoybean Products Processed Soy Protein Products Roasted Soybeans FullFat and Defatted Flours Baked Soybeans Soy Grits Soy Sprouts SoyHypocotyls Soy Milk Soybean Meal Specialty Soy Foods/Ingredients SoyMilk Soy Milk Soy Protein Isolates Tofu Soy Protein Concentrates TempehTextured Soy Proteins Miso Textured Flours and Concentrates Soy SauceTextured Concentrates Hydrolyzed Vegetable Protein Textured IsolatesWhipping Protein ^(a)See Soy Protein Products: Characteristics,Nutritional Aspects and Utilization (1987). Soy Protein Council.

“Processing” refers to any physical and chemical methods used to obtainthe products listed in Table 1A and includes, but is not limited to,heat conditioning, flaking and grinding, extrusion, solvent extraction,or aqueous soaking and extraction of whole or partial seeds.Furthermore, “processing” includes the methods used to concentrate andisolate soy protein from whole or partial seeds, as well as the varioustraditional Oriental methods in preparing fermented soy food products.Trading Standards and Specifications have been established for many ofthese products (see National Oilseed Processors Association Yearbook andTrading Rules 1991-1992). Products referred to as being “high protein”or “low protein” are those as described by these StandardSpecifications. “NSI” refers to the Nitrogen Solubility Index as definedby the American Oil Chemists' Society Method Ac4 41. “KOH NitrogenSolubility” is an indicator of soybean meal quality and refers to theamount of nitrogen soluble in 0.036 M KOH under the conditions asdescribed by Araba and Dale [(1990) Poult. Sci. 69:76-83]. “White”flakes refer to flaked, dehulled cotyledons that have been defatted andtreated with controlled moist heat to have an NSI of about 85 to 90.This term can also refer to a flour with a similar NSI that has beenground to pass through a No. 100 U.S. Standard Screen size. “Cooked”refers to a soy protein product, typically a flour, with an NSI of about20 to 60. “Toasted” refers to a soy protein product, typically a flour,with an NSI below 20. “Grits” refer to defatted, dehulled cotyledonshaving a U.S. Standard screen size of between No. 10 and 80. “SoyProtein Concentrates” refer to those products produced from dehulled,defatted soybeans by three basic processes: acid leaching (at about pH4.5), extraction with alcohol (about 55-80%), and denaturing the proteinwith moist heat prior to extraction with water. Conditions typicallyused to prepare soy protein concentrates have been described by Pass[(1975) U.S. Pat. No. 3,897,574; Campbell et al., (1985) in New ProteinFoods, ed. by Altschul and Wilcke, Academic Press, Vol. 5, Chapter 10,Seed Storage Proteins, pp 302-338]. “Extrusion” refers to processeswhereby material (grits, flour or concentrate) is passed through ajacketed auger using high pressures and temperatures as a means ofaltering the texture of the material. “Texturing” and “structuring”refer to extrusion processes used to modify the physical characteristicsof the material. The characteristics of these processes, includingthermoplastic extrusion, have been described previously [Atkinson (1970)U.S. Pat. No. 3,488,770, Horan (1985) In New Protein Foods, ed. byAltschul and Wilcke, Academic Press, Vol. 1A, Chapter 8, pp 367-414].Moreover, conditions used during extrusion processing of complexfoodstuff mixtures that include soy protein products have been describedpreviously [Rokey (1983) Feed Manufacturing Technology III, 222-237;McCulloch, U.S. Pat. No. 4,454,804].

TABLE 1B Generalized Steps for Soybean Oil and Byproduct ProductionProcess Impurities Removed and/or Step Process By-Products Obtained # 1soybean seed # 2 oil extraction meal # 3 Degumming lecithin # 4 alkalior physical gums, free fatty acids, refining pigments # 5 water washingsoap # 6 Bleaching color, soap, metal # 7 (hydrogenation) # 8(winterization) stearine # 9 Deodorization free fatty acids,tocopherols, sterols, volatiles # 10  oil products

More specifically, soybean seeds are cleaned, tempered, dehulled, andflaked, thereby increasing the efficiency of oil extraction. Oilextraction is usually accomplished by solvent (e.g., hexane) extractionbut can also be achieved by a combination of physical pressure and/orsolvent extraction. The resulting oil is called crude oil. The crude oilmay be degummed by hydrating phospholipids and other polar and neutrallipid complexes that facilitate their separation from the nonhydrating,triglyceride fraction (soybean oil). The resulting lecithin gums may befurther processed to make commercially important lecithin products usedin a variety of food and industrial products as emulsification andrelease (i.e., antisticking) agents. Degummed oil may be further refinedfor the removal of impurities (primarily free fatty acids, pigments andresidual gums). Refining is accomplished by the addition of a causticagent that reacts with free fatty acid to form soap and hydratesphosphatides and proteins in the crude oil. Water is used to wash outtraces of soap formed during refining. The soapstock byproduct may beused directly in animal feeds or acidulated to recover the free fattyacids. Color is removed through adsorption with a bleaching earth thatremoves most of the chlorophyll and carotenoid compounds. The refinedoil can be hydrogenated, thereby resulting in fats with various meltingproperties and textures. Winterization (fractionation) may be used toremove stearine from the hydrogenated oil through crystallization undercarefully controlled cooling conditions. Deodorization (principally viasteam distillation under vacuum) is the last step and is designed toremove compounds which impart odor or flavor to the oil. Other valuablebyproducts such as tocopherols and sterols may be removed during thedeodorization process. Deodorized distillate containing these byproductsmay be sold for production of natural vitamin E and other high-valuepharmaceutical products. Refined, bleached, (hydrogenated, fractionated)and deodorized oils and fats may be packaged and sold directly orfurther processed into more specialized products. A more detailedreference to soybean seed processing, soybean oil production, andbyproduct utilization can be found in Erickson, Practical Handbook ofSoybean Processing and Utilization, The American Oil Chemists' Societyand United Soybean Board (1995). Soybean oil is liquid at roomtemperature because it is relatively low in saturated fatty acids whencompared with oils such as coconut, palm, palm kernel, and cocoa butter.

Plant and microbial oils containing PUFAs that have been refined and/orpurified can be hydrogenated, thereby resulting in fats with variousmelting properties and textures. Many processed fats (including spreads,confectionary fats, hard butters, margarines, baking shortenings, etc.)require varying degrees of solidity at room temperature and can only beproduced through alteration of the source oil's physical properties.This is most commonly achieved through catalytic hydrogenation.

Hydrogenation is a chemical reaction in which hydrogen is added to theunsaturated fatty acid double bonds with the aid of a catalyst such asnickel. For example, high oleic soybean oil contains unsaturated oleic,linoleic, and linolenic fatty acids, and each of these can behydrogenated. Hydrogenation has two primary effects. First, theoxidative stability of the oil is increased as a result of the reductionof the unsaturated fatty acid content. Second, the physical propertiesof the oil are changed because the fatty acid modifications increase themelting point resulting in a semi-liquid or solid fat at roomtemperature.

There are many variables which affect the hydrogenation reaction, whichin turn alter the composition of the final product. Operating conditionsincluding pressure, temperature, catalyst type and concentration,agitation, and reactor design are among the more important parametersthat can be controlled. Selective hydrogenation conditions can be usedto hydrogenate the more unsaturated fatty acids in preference to theless unsaturated ones. Very light or brush hydrogenation is oftenemployed to increase stability of liquid oils. Further hydrogenationconverts a liquid oil to a physically solid fat. The degree ofhydrogenation depends on the desired performance and meltingcharacteristics designed for the particular end product. Liquidshortenings (used in the manufacture of baking products, solid fats andshortenings used for commercial frying and roasting operations) and basestocks for margarine manufacture are among the myriad of possible oiland fat products achieved through hydrogenation. A more detaileddescription of hydrogenation and hydrogenated products can be found inPatterson, H. B. W., Hydrogenation of Fats and Oils: Theory andPractice. The American Oil Chemists' Society (1994).

Hydrogenated oils have become somewhat controversial due to the presenceof trans-fatty acid isomers that result from the hydrogenation process.Ingestion of large amounts of trans-isomers has been linked withdetrimental health effects including increased ratios of low density tohigh density lipoproteins in the blood plasma and increased risk ofcoronary heart disease.

EXAMPLES

The present invention is further defined in the following Examples, inwhich parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions. Thus, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “A” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Example 1 Expression of Yarrowia lipolytica DGAT Genes in Saccharomycescerevisiae

The DGAT1 gene (SEQ ID NO:1) of Yarrowia lipolytica was excised fromplasmid vector pYDA1 (SEQ ID NO:2) by restriction digestion with NcoIand NotI. The ends of DNA fragment were completely filled in using T4DNA polymerase (Promega, Madison, Wis., USA) and ligated into the uniqueNot I site of pY75 (SEQ ID NO:3). Prior to its use for cloning the pY75vector had been linearized with NotI, filled in with T4 DNA polymeraseand dephosphorylated with shrimp alkaline phosphatase (NEB, Beverly,Mass., USA). Plasmid DNA was isolated using standard techniques andrestriction digests with EcoRI were conducted to identify plasmid clonesin which the start codon was in proximity to the 3′ end of the GPDpromoter in pY75 (sense orientation of the DGAT1 gene). This plasmid ishenceforth referred to as pY75 YL DGAT1 (SEQ ID NO:4). The constructionof pYDA1 is described in PCT Publication No. WO 2006/052914, which ishereby incorporated as reference.

The yeast episomal plasmid (YEp)-type vector pRS425 (SEQ ID NO:5)(Christianson et al., Gene 110:119-122 (1992)) contains sequences fromthe Saccharomyces cerevisiae 2 micron endogenous plasmid, a LEU2selectable marker and sequences based on the backbone of amultifunctional phagemid, pBluescript II SK(+). The Saccharomycescerevisiae strong, constitutive glyceraldehyde-3-phosphate dehydrogenase(GPD) promoter was cloned between the SacII and SpeI sites of pRS425 inthe same way as described by Jia et al. (Physiol. Genomics 3:83-92(2000)) to produce pGPD-425 (SEQ ID NO:6). A NotI site was introducedinto the BamHI site of pGPD-425, thus giving a NotI site flanked byBamHI sites, and this plasmid was called pY75 (SEQ ID NO:3) The DGAT2gene was PCR amplified from the genome of Yarrowia lipolytica (ATCCAccession No. 20362) as follows. Yeast cells were grown on solid YPDmedium for 72 h. Cells were resuspended in 200 μL of DNA extractionbuffer (100 mM Tris pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.1% Triton X-100)and supplemented with 2-5 glass beads (3 mm diameter) and approximately0.1 g of glass beads (0.5 mm diameter). The yeast cell suspension wasmixed vigorously using a vortex mixer and incubated at 75° C. for 25min. The lysate was cooled to room temperature and cleared bycentrifugation.

The following two oligonucleotide primers were used to generate a PCRfragment of approximately 1600 bp:

oYLDGAT2-1: (SEQ ID NO: 7) GCGGCCGCATGACTATCGACTCACAATACTACAAGT, andoYLDGAT2-2: (SEQ ID NO: 8) GCGGCCGCTTACTCAATCATTCGGAACTCTGGGGCT.Briefly, a PCR reaction mixture (100 μL) containing 2.5 mM MgCl₂, 2 mMdNTPs, 10 mM Tris/HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P40, 1 μM ofoYLDGAT2-1 (SEQ ID NO:7) and oYLDGAT2-2 (SEQ ID NO:8), 10 U Taqpolymerase (Fermentas, Hanover, Md.), and 2 μL of yeast lysate wascreated. The PCR mixture was divided into four 25 μL aliquots andamplification was carried out for 35 cycles, each comprising 45 sec at94° C., 45 sec at the respective annealing temperature, and 1 min at 72°C. PCR products were gel-purified and cloned into pGEM T-easy (Promega)using manufacturer instructions.

Ten independent plasmid clones were completely sequenced. The consensussequence of this analysis is set forth as SEQ ID NO:9. This DNA sequencediffers from the DGAT2 sequence disclosed in PCT Publication No. WO2005/003322 at two different nucleotide positions. The difference in DNAsequence affects nt 448 and nt 672 of the DGAT2 open reading frame. Theformer nt difference changes the predicted amino acid sequence of theDGAT protein. It replaces a serine found in the DGAT sequence disclosedin PCT Publication No. WO 2005/003322 with a threonine residue. Thesecond sequence difference does not change the amino acid sequence fromthe one disclosed in PCT Publication No. WO 2005/003322. The differencebetween the Yarrowia lipolytica DGAT2 sequence disclosed herein and thatof PCT Publication No. WO 2005/003322 can be attributed to the differentYarrowia lipolytica strains that were used for DGAT2 gene isolation. Thepredicted amino acid sequence of the DGAT protein of strain ATCCAccession No. 20362 is set forth as SEQ ID NO:10.

The DGAT gene (SEQ ID NO:9) was excised as a Not I restriction fragmentfrom the pGEM T-easy vector and ligated to NotI linearized,dephosphorylated DNA of pY75 (SEQ ID NO:3). Plasmid DNA was isolatedform recombinant clones and restriction digestion with SacI and Padallowed to identify clones in which the start codon of the DGAT2 genewas in proximity to the 3′ end of the GPD promoter in pY75 (senseorientation of the DGAT2 gene). This plasmid is henceforth referred toas pY75 YL DGAT2 (SEQ ID NO:11).

Plasmid DNA of pY75 YL DGAT1 (SEQ ID NO:4) and the empty pY75 vectorwere transformed into the Saccharomyces cerevisiae stain INVSC1(Invitrogen, USA) using standard methods (Gietz, R. Daniel; Woods, RobinA., Meth. Enzymol. 350:87-96 (2002)). Recombinant yeast colonies wereselected on DOBA media supplemented with CSM-leu (Qbiogene, Carlsbad,Calif.). Five 50 mL cultures of DOBA media supplemented with CSM-leuwere inoculated with five independently generated colonies and grown and30° C. for 72 h. Cells were harvested by centrifugation and resuspendedin medium identical to the DOBA medium described above with theexception that ammonium sulfate as nitrogen source was omitted. Cultureswere grown for additional 60 h, cells were harvested by centrifugation.Cells were frozen on dry ice and lyophilized.

Total fatty acid content of each yeast cell sample was measured intriplicates as follows. Approximately 5-15 mg of yeast powder wereweighed into the bottom of a 13×100 mm glass culture tube with screw capand Teflon seal. 5 μL of a stock solution of 17:0 TAG (10 mg/mL intoluene) was added followed by addition of 500 μL 5% sulfuric acid inmethanol (anhydrous). Samples were incubated at 95° C. for 1.5 h.Subsequently, tubes were allowed to cool to room temperature after which1 mL of 1 M sodium chloride was added followed by mixing. 1 mL ofheptane was added, contents were mixed and samples were spun briefly tomediate phase separation. Approximately 500 μL of the organic phase wastransferred to a GC vial. Fatty acid methyl esters were analyzed by gaschromatography. 4 μL of heptane extract were analyzed on Hewlett-Packard6890 Gas Chromatograph fitted with an Omegawax 320 fused silicacapillary column (Supelco Inc., Catalog No. 24152). The oven temperaturewas programmed to hold at 220° C. for 2.7 min, increase to 240° C. at 20C/min and then hold for an additional 2.3 min. Carrier gas was suppliedby a Whatman hydrogen generator. Retention times were compared to thosefor methyl esters of standards commercially available (Nu-Chek Prep,Inc. catalog #U-99-A).

Plasmid DNA of pY75 YL DGAT2 (SEQ ID NO:11) and the empty pY75 vectorwere transformed into the Saccharomyces cerevisiae strain INVSC1 andtotal fatty acid content of recombinant yeast cultures was analyzed asdescribed previously. The findings related to over expression of bothDGAT genes in yeast are summarized in TABLE 2.

TABLE 2 Total Fatty Acid Content of Saccharomyces cerevisea Culturesaverage stdv % % % % FAME FAME FAME palmitic palmitoleic stearic oleic(% (% (% acid acid acid acid DCW) stdv DCW) DCW) pY75 YL 19.1 38.4 7.534.9 12.8 2.2 DGAT1 19.4 38.8 7.4 34.3 12.3 0.4 19.2 38.4 7.6 34.8 12.20.4 19.3 38.6 7.5 34.6 11.5 0.1 19.1 38.3 7.7 34.9 10.9 0.6 11.9 0.8pY75 17.9 37.8 7.9 36.4 10.7 1.0 18.2 38.2 7.8 35.8 9.7 0.6 17.9 41.06.8 34.2 8.7 0.3 17.2 40.5 6.9 35.4 8.7 0.6 18.1 41.1 6.9 33.9 8.5 0.29.3 0.9 pY75 YL 31.8 34.0 14.2 20.0 17.1 0.2 DGAT2 31.4 33.1 14.8 20.615.9 0.6 30.7 32.8 14.7 21.8 13.6 1.1 28.9 34.2 13.5 23.5 12.4 1.4 29.234.1 13.5 23.1 11.8 1.7 14.2 2.3 pY75 19.7 37.0 9.6 33.7 7.0 0.4 20.036.4 9.8 33.7 6.8 0.0 19.6 37.0 9.6 33.8 6.6 0.6 19.7 37.1 9.5 33.6 6.40.2 19.3 36.9 9.7 34.1 6.4 0.3 6.6 0.2

TABLE 2 shows that there is a significant increase of total fatty acidsin yeast cells harboring the pY75 YL DGAT1 (SEQ ID NO:4) compared tocells that only contain the empty pY75 plasmid. The average fatty aciddry cell weigt (DCW) percentage of five independent cultures is 11.9%compared to 9.3% for vector controls grown under identical conditions.In summary, there is a 28% increase in total fatty acid production.Moreover, there is a slight alteration in the fatty acid profileassociated with YL DGAT1 expression characterized by an increase inpalmitic acid.

Constitutive expression of YL DGAT2 under nitrogen starvation increasedtotal fatty acid content by 110% compared to a vector control grownunder identical conditions. Total fatty acid content of the vector onlycontrol was 6.6% whereas the average fatty acid content of the YL DGAT2transformants was 14.2%. The fatty acid profile changed as result of YLDGAT2 expression. Palmitic acid content increased significantlyaccompanied by a moderate decrease in palmitoleic. In addition, stearicacid content increased significantly accompanied by a clear decrease inoleic content. Taken together the results show that YL DGATsover-expression in yeast under conditions of increased carbon/nitrogenratios lead to increased fatty acid accumulation. The overexpressed YLDGAT enzymes are able to augment endogenous DGAT activity inSaccharomyces cerevisiae. Similar experiments were repeated with bothDGAT genes two more times. A difference in fatty acid content between YLDGAT culture and vector control could be observed every time and was atleast 8% and 14.3% for YL DGAT1 (SEQ ID NO:1) and YL DGAT2 (SEQ IDNO:9), respectively.

Example 2 Cloning the Yarrowia lipolytica DGAT1 and DGAT2 into Yarrowialipolytica Expression Vectors

The present Example describes the generation of pFBAIN-YDG1 andpFBAIN-YDG2, comprising a chimeric FBAINm::YDGAT1::PEX20 gene and achimeric FBAINm::YDGAT2::PEX20 gene, respectively (FIGS. 1A and 1B).These were designed for overexpression of the DGAT1 and DGAT2 inYarrowia lipolytica.

Oligonucleotides YDGAT1-F (SEQ ID NO:12) and YDGAT-R (SEQ ID NO:13) weredesigned and synthesized to allow amplification of the DGAT1 ORF fromYarrowia lipolytica genomic DNA (isolated from strain ATCC Accession No.20362, purchased from the American Type Culture Collection (Rockville,Md.)), while oligonucleotides YDGAT2-F (SEQ ID NO:14) and YDGAT2-R (SEQID NO:15) were designed and synthesized to allow the amplification ofthe DGAT2 ORF.

The PCR reactions, with Yarrowia lipolytica genomic DNA as template,were individually carried out in a 50 μL total volume comprising: 1 μLeach of 20 μM forward and reverse primers, 1 μL genomic DNA (100 ng), 10μL 5×PCR buffer, 1 μL dNTP mix (10 μM each), 35 μL water and 1 μLPhusion polymerase (New England Biolabs, Inc., Ipswich, Mass.).Amplification was carried out at 98° C. for 1 min, followed by 30 cyclesat 98° C. for 10 sec, 55° C. for 10 sec, and 72° C. for 30 sec, followedby a final elongation cycle at 72° C. for 5 min. A 1603 bp DNA fragment(SEQ ID NO:16) and a 1567 bp fragment (SEQ ID NO:17) were generated thatcontained the DGAT1 and DGAT2 ORFs, respectively.

The PCR fragments were purified with Qiagen PCR purification kitsfollowing the manufacturer's protocol. Purified DNA samples weredigested with NcoI and NotI, purified with a Qiagen reaction clean-upkit, and then directionally ligated with NcoI/NotI digested pFBAIN-MOD-1(FIG. 1C; SEQ ID NO:18). Specifically, the ligation reaction contained:10 μL 2× ligation buffer, 1 μL T4 DNA ligase (Promega), 4 μL (˜300 ng)of either the 1600 bp fragment (i.e., DGAT1; SEQ ID NO:16) or the 1564bp fragment (i.e., DGAT2; SEQ ID NO:17) and 1 μL pFBAIN-MOD-1 (˜150 ng).The reaction mixtures were incubated at room temperature for 2 h andused to transform E. coli Top10 competent cells (Invitrogen). PlasmidDNA from transformants was recovered using a Qiagen Miniprep kit.Correct clones were identified by restriction mapping and the finalconstructs were designated “pFBAIN-YDG1” and “pFBAIN-YDG2”,respectively.

Thus, pFBAIN-YDG1 (FIG. 1A; SEQ ID NO:19) thereby contained thefollowing components:

TABLE 3 Components of Plasmid pFBAIN-YDG1 (SEQ ID NO: 19) RE Sites AndNucleotides Within SEQ Description of Fragment and ID NO: 19 ChimericGene Components BgIII-BsiWI FBAINm::YDG1::PEX20, comprising: (6040-301) FBAINm: Yarrowia lipolytica FBAINm promoter (PCT Publication No. WO2005/049805) YDG1: Y. lipolytica DGAT1 ORF (SEQ ID NO: 16) Pex20: Pex20terminator sequence of Yarrowia Pex20 gene (GenBank Accession No.AF054613) PacI-BgIII Yarrowia URA3 (GenBank Accession No. AJ306421)(4533-6040) (3123-4487) Yarrowia autonomous replicating sequence 18(ARS18; GenBank Accession No. M91600 and No. A17608) (2464-2864) f1origin (1424-2284) ampicillin-resistance gene (Amp^(R)) for selection inE. coli  (474-1354) ColE1 plasmid origin of replicationPlasmid pFBAIN-YDG2 (FIG. 1B; SEQ ID NO:20) contained componentsidentical to those of pFBAIN-YDG1, with the exception that the Yarrowialipolytica DGAT2 ORF (SEQ ID NO:17; identified as YDG2 on FIG. 1B) waspresent instead of the Yarrowia lipolytica DGAT1 ORF in pFBAIN-YDG1.

The term “FBAINm promoter” or “FBAINm promoter region” is a modifiedversion of the FBAIN promoter (infra), wherein FBAINm has a 52 bpdeletion between the ATG translation initiation codon and the intron ofthe FBAIN promoter (thereby including only 22 amino acids of theN-terminus) and a new translation consensus motif after the intron.Furthermore, while the FBAIN promoter generates a fusion protein whenfused with the coding region of a gene to be expressed, the FBAINmpromoter does not generate such a fusion protein. The FBAIN promoterrefers to the 5′ upstream untranslated region in front of the ‘ATG’translation initiation codon of the Yarrowia lipolyticafructose-bisphosphate aldolase enzyme (E.C. 4.1.2.13) encoded by thefba1 gene and that is necessary for expression, plus a portion of 5′coding region that has an intron of the fba1 gene. These promoters aredescribed in detail in PCT Publication No. WO 2005/049805 and U.S. Pat.No. 7,202,356, which are hereby incorporated by reference in theirentirety.

Example 3 Overexpression of Yarrowia lipolytica DGAT1 And DGAT2 Genes inYarrowia lipolytica Strain Y2224

The present Example describes increased fatty acid content, andmodification to the relative abundance of each fatty acid species, inYarrowia lipolytica strain Y2224 that was transformed to co-expresseither the Yarrowia lipolytica DGAT1 (SEQ ID NO:16) or the Yarrowialipolytica DGAT2 (SEQ ID NO:17). Strain Y2224 is a FOA resistant mutantfrom an autonomous mutation of the Ura3 gene of wild type Yarrowiastrain ATCC Accession No. 20362.

Generation of Strain Y2224:

Strain Y2224 was isolated in the following manner: Yarrowia lipolyticaATCC Accession No. 20362 cells from a YPD agar plate (1% yeast extract,2% bactopeptone, 2% glucose, 2% agar) were streaked onto a minimal mediaplate (75 mg/L each of uracil and uridine, 6.7 g/L YNB with ammoniasulfate, without amino acid, and 20 g/L glucose) containing 250 mg/L5-FOA (5-fluorouracil-6-carboxylic acid monohydrate; Zymo Research).Plates were incubated at 28° C. and four of the resulting colonies werepatched separately onto minimal media (MM) plates containing 200 mg/mL5-FOA and MM plates lacking uracil and uridine to confirm uracil Ura3auxotrophy.

Transformation of Strain Y2224:

A clone of pFBAIn-YDG1, a clone of pFBAIn-YDG2 and control plasmidpFBAIN-MOD-1 were transformed into Yarrowia lipolytica strain Y2224 asdescribed below.

Transformation of Yarrowia lipolytica was performed according to themethod of Chen, D. C. et al. (Appl. Microbiol Biotechnol. 48(2):232-235(1997)), unless otherwise noted. Briefly, Yarrowia was streaked onto aYPD agar plate and grown at 30° C. for approximately 18 h. Several largeloopfuls of cells were scraped from the plate and resuspended in 1 mL oftransformation buffer containing: 2.25 mL of 50% PEG, average MW 3350;0.125 mL of 2 M lithium acetate, pH 6.0; 0.125 mL of 2 M DTT; and 50 μgsheared salmon sperm DNA. Then, approximately 500 ng of linearizedplasmid DNA was incubated in 100 μL of resuspended cells, and maintainedat 39° C. for 1 h with vortex mixing at 15 min intervals.

The cells from each transformation were plated onto minimal media (MM)plates lacking uracil (0.17% yeast nitrogen base (DIFCO Laboratories,Detroit, Mich.) without ammonium sulfate or amino acids, 2% glucose,0.1% proline, pH 6.1, 20 g/L agar) and maintained at 30° C. for 2 days.Three transformants from each transformation plate were used toinoculate individual 25 mL culture in MM medium (0.17% yeast nitrogenbase (DIFCO Laboratories) without ammonium sulfate or amino acids, 2%glucose, 0.1% proline, pH 6.1). Each culture was allowed to grow for 2days at 30° C., then switched into 25 mL of high glucose medium (“HGmedium”, comprising 80 g/L glucose, 27 g/L K₂HPO₄, 6.3 g/L KH₂PO₄, pH˜7.5) and allowed to grow for 5 days at 30° C.

Lipid Analysis:

Total lipids were extracted, and fatty acid methyl esters (FAMEs) wereprepared by trans-esterification, and subsequently analyzed with aHewlett-Packard 6890 GC.

More specifically, for fatty acid analysis, cells were collected bycentrifugation and lipids were extracted as described in Bligh, E. G. &Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)). Fatty acidmethyl esters were prepared by transesterification of the lipid extractwith sodium methoxide (Roughan, G. and Nishida I., Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μL of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μL hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Based on the above analyses, lipid content and composition wasdetermined in transformant strains of Y2224, comprising pFBAIn-YDG1,pFBAIn-YDG2 and pFBAIN-MOD-1 (control), respectively, as shown below inTABLE 4. Three independent transformants of each strain were analyzed,while the average results are shown in the rows highlighted in grey.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid) and 18:2 (LA); and the composition of each ispresented as a % of the total fatty acids.

“% FAME/DCW” represents the percent fatty acid methyl ester/dry cellweight. Dry cell weight was determined by collecting cells from 10 mL ofculture via centrifugation, washing the cells with water once to removeresidue medium, drying the cells in a vacuum oven at 80° C. overnight,and weighing the dried cells. The total amount of fatty acid methylesters in a sample was determined by comparing the areas of all peaks inthe GC profile with the peak area of an added known amount of internalstandard C15:0 fatty acid.

GC analyses showed that there was a significant increase of total fattyacids in cells carrying pFBAIn-YDG1, as compared to cells carryingpFBAIn-MOD-1. The average fatty acid increased from 21.39% FAME/DCW inthe control to 23.70% FAME/DCW in cells expressing YDGAT1 (i.e., a 10.8%increase). Furthermore, there was also an increase in the amount ofC16:1 and C18:1 fatty acids and a decrease of C18:2 fatty acid (TABLE4).

Cells carrying pFBAIn-YDG2 also had a large increase in total fatty acidcontent relative to the control, resulting in an average of 27.32%FAME/DCW (representing an increase of 27.7%). The distribution of fattyacid species also changed significantly. Specifically, the amount ofC18:0 and C18:1 was increased, whereas the amount of C16:0, C16:1 andC18:2 decreased.

Collectively, these results demonstrate that overexpression of theYarrowia lipolytica DGAT1 or DGAT2 impacts both total lipid content andthe relative abundance of each fatty acid species.

Example 4

Expression of Yarrowia lipolytica DGAT genes in Arabidopsis Seed Abinary vector suitable for agrobacterium-mediated transformation wasgenerated as follows. Various restriction sites were added, through anumber of cloning steps, to the ends of the Bcon/NotI/Phas3′ cassettefrom KS123 (SEQ ID NO:21), which was previously described in PCTPublication No. WO 02/008269 (the contents of which are herebyincorporated by reference). Briefly, a DNA fragment (cal a24-4; SEQ IDNO:22) was amplified from plasmid CalFad2-2 (described in PCTPublication No. WO 01/12800) using primers oCal-15 (SEQ ID NO:23) andoCal-6 (SEQ ID NO:24). DNA fragment cal a24-4 (SEQ ID NO:22) wasdigested with BglII and BamHI and cloned into the BamHI site of pKS123to give pKR53B (SEQ ID NO:25). The XbaI/SbtI fragment of pKR53B,containing the Bcon/NotI/Phas3′ cassette was cloned into the XbaI/SbtIfragment of pKR72 (ATCC Accession No. PTA-6019; SEQ ID NO:26) containingthe bacterial hygromycin phosphotransferase gene, to give pKR85 (SEQ IDNO:27). The features of pKR72 are as follows. A starting plasmid pKR72(ATCC Accession No. PTA-6019; SEQ ID NO:26), a derivative of pKS123which was previously described in PCT Publication No. WO 02/008269 (thecontents of which are hereby incorporated by reference), contains thehygromycin B phosphotransferase gene (HPT) (Gritz, L. and Davies, J.,Gene 25:179-188 (1983)), flanked by the T7 promoter and transcriptionterminator (T7prom/hpt/T7term cassette), and a bacterial origin ofreplication (ori) for selection and replication in bacteria (e.g., E.coli). In addition, pKR72 also contains the hygromycin Bphosphotransferase gene, flanked by the 35S promoter (Odell et al.,Nature 313:810-812 (1985)) and NOS3′ transcription terminator (Depickeret al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3′ cassette)for selection in plants such as soybean. pKR72 also contains a NotIrestriction site, flanked by the promoter for the a′ subunit ofβ-conglycinin (Beachy et al., EMBO J. 4:3047-3053 (1985)) and the 3′transcription termination region of the phaseolin gene (Doyle et al., J.Biol. Chem. 261:9228-9238 (1986)), thus allowing for strongtissue-specific expression in the seeds of soybean of genes cloned intothe NotI site.

The Bcon/NotI/Phas3′ cassette was amplified from plasmid pKR85 (SEQ IDNO:27) using primers oKR85-1 (SEQ ID NO:28) and oKR85-2 (SEQ ID NO:29)and the resulting DNA fragment was cloned into PCR-Script® (Stratgene)following the manufacture's protocol, to give pPCR85 (SEQ ID NO:30).

The EcoRI/BglII fragment of pPCR85, containing the Bcon/NotI/Phas3′cassette was cloned into the EcoRI/BamHI fragment of plasmid pZS199 (PCTPublication No. WO 93/11245; also U.S. Pat. No. 5,952,544 which waspublished on Jun. 10, 1993; the disclosures of which are herebyincorporated by reference), containing the Arabidopsis binary vectorbackbone to produce pKR91 (SEQ ID NO:31).

The Bcon/NotI/Phas3′ cassette was removed from pKR91 by digestion withAscI and the re-ligated binary vector containing a unique AscI cloningsite was produced called pKR92 (SEQ ID NO:32).

Construction of pKR92 YL DGAT2:

The construction of expression plasmid KS362 is described in Example 5.An expression cassette which harbors the YL DGAT2 gene, fused tobetaconglycinin promoter and the phaseolin terminator and DsRed genefused to Kti promoter and terminator sequences was excised from KS362 asa 6.4 kb AscI fragment. This DNA was ligated to AscI linearized,dephosphorylated pKR92 vector DNA to give pKR92 YL DGAT2 (SEQ ID NO:33).

Construction of pKR92 YL DGAT1/YL DGAT2:

The construction of expression plasmid KS364 is described in Example 5.An expression cassette in which YL DGAT1 (SEQ ID NO:1) and YL DGAT2 (SEQID NO:9) genes are fused to identical sequence of the phaseolinterminator and to glycinin 1 and betaconglycinin promoters respectivelywas excised from KS364 as a 7 kb AscI fragment. This DNA was ligated toAscI linearized, dephosphorylated pKR92 vector DNA to give pKR92 YLDGAT1/YL DGAT2 (SEQ ID NO:34)

Generation and Analysis of Transgenic Arabidospis Lines:

Plasmid DNA of pKR92 YL DGAT2 and pKR92 YL DGAT1/YL DGAT2 was introducedinto Agrobacterium tumefaciens NTL4 (Luo et al, Molecular Plant-MicrobeInteractions 14(1):98-103 (2001)) by electroporation. Briefly, 1 μgplasmid DNA was mixed with 100 μL of electro-competent cells on ice. Thecell suspension was transferred to a 100 μL electro oration curette (1mm gap width) and electro orated using a BIORAD electro orator set to 1kV, 400Ω and 25 μF. Cells were transferred to 1 mL LB medium andincubated for 2 h at 30° C. Cells were plated onto LB medium containing50 μg/mL kanamycin. Plates were incubated at 30° C. for 60 h.Recombinant agrobacterium cultures (500 mL LB, 50 μg/mL kanamycin) wereinoculated from single colonies of transformed agrobacterium cells andgrown at 30° C. for 60 h. Cells were harvested by centrifugation(5000×g, 10 min) and resuspended in 1 L of 5% (W/V) sucrose containing0.05% (V/V) Silwet. Arabidopsis plants were grown in soil at a densityof 30 plants per 100 cm² pot in metromix 360 soil mixture for 4 weeks(22° C., permanent light, 100 μE m⁻² s⁻¹). Plants were repeatedly dippedinto the agrobacterium suspension harboring the binary vectors and keptin a dark, high humidity environment for 24 h. Plants were grown forfour to five weeks under standard plant growth conditions describedabove and plant material was harvested and dried for one week at ambienttemperatures in paper bags. Seeds were harvested using a 0.425 mm meshbrass sieve.

Cleaned Arabidopsis seeds (2 g, corresponding to about 100,000 seeds)were sterilized by washes in 45 mL of 80% ethanol, 0.01% triton X-100,followed by 45 mL of 30% (V/V) household bleach in water, 0.01% tritonX-100 and finally by repeated rinsing in sterile water. Aliquots of20,000 seeds were transferred to square plates (20×20 cm) containing 150mL of sterile plant growth medium comprised of 0.5×MS salts, 1.0% (W/V)sucrose, 0.05 MES/KOH (pH 5.8), 200 μg/mL timentin, and 50 μg/mLkanamycin solidified with 10 g/L agar. Homogeneous dispersion of theseed on the medium was facilitated by mixing the aqueous seed suspensionwith an equal volume of melted plant growth medium. Plates wereincubated under standard growth conditions for ten days.Kanamycin-resistant seedlings were transferred to plant growth mediumwithout selective agent and grown to maturity for 8-10 weeks (22° C.,permanent light dark, 100-200 g m⁻² s⁻¹). Plants were grown in flatswith 36 inserts. In every flat at least six untransformed wild typecontrol plants were grown next to approximately thirty T2 plants. Seedswere harvested from individual plants and seed oil content was measuredby NMR.

NMR Based Analysis of Seed Oil Content:

Seed oil content was determined using a Maran Ultra NMR analyzer(Resonance Instruments Ltd, Whitney, Oxfordshire, UK). Samples (eitherindividual soybean seed or batches of Arabidopsis seed ranging in weightbetween 5 and 200 mg) were placed into pre-weighed 2 mL polypropylenetubes (Corning Inc, Corning N.Y., USA; Part no. 430917) previouslylabeled with unique bar code identifiers. Samples were then placed into96 place carriers and processed through the following series of steps byan Adept Cobra 600 SCARA robotic system.

-   -   1. pick up tube (the robotic arm was fitted with a vacuum pickup        devise)    -   2. read bar code    -   3. expose tube to antistatic device (ensured that Arabidopsis        seed were not adhering to the tube walls)    -   4. weigh tube (containing the sample), to 0.0001 g precision.    -   5. NMR reading; measured as the intensity of the proton spin        echo 1 msec after a 22.95 MHz signal had been applied to the        sample (data was collected for 32 NMR scans per sample)    -   6. return tube to rack    -   7. repeat process with next tube        Bar codes, tubes weights and NMR readings were recorded by a        computer connected to the system. Sample weight was determined        by subtracting the polypropylene tube weight from the weight of        the tube containing the sample.

Seed oil content of soybeans seed was calculated as follows:

${\% \mspace{14mu} {oil}\mspace{14mu} \left( {\% \mspace{14mu} {wt}\mspace{14mu} {basis}} \right)} = \frac{\left. {\left( {{NMR}\mspace{14mu} {signal}\text{/}{sample}\mspace{14mu} {wt}\mspace{14mu} (g)} \right) - 70.58} \right)}{351.45}$

Calibration parameters were determined by precisely weighing samples ofsoy oil (ranging from 0.0050 to 0.0700 g at approximately 0.0050 gintervals; weighed to a precision of 0.0001 g) into Corning tubes (seeabove) and subjecting them to NMR analysis. A calibration curve of oilcontent (% seed wt basis; assuming a standard seed weight of 0.1500 g)to NMR value was established.

The relationship between seed oil contents measured by NMR and absoluteoil contents measured by classical analytical chemistry methods wasdetermined as follows. Fifty soybean seed, chosen to have a range of oilcontents, were dried at 40° C. in a forced air oven for 48 h. Individualseeds were subjected to NMR analysis, as described above, and were thenground to a fine powder in a GenoGrinder (SPEX Centriprep (Metuchen,N.J., U.S.A.); 1500 oscillations per minute, for 1 minute). Aliquots ofbetween 70 and 100 mg were weighed (to 0.0001 g precision) into 13×100mm glass tubes fitted with Teflon® lined screw caps; the remainder ofthe powder from each bean was used to determine moisture content, byweight difference after 18 h in a forced air oven at 105° C. Heptane (3mL) was added to the powders in the tubes and after vortex mixingsamples were extracted, on an end-over-end agitator, for 1 h at roomtemperature. The extracts were centrifuged, 1500×g for 10 min, thesupernatant decanted into a clean tube and the pellets were extractedtwo more times (1 h each) with 1 mL heptane. The supernatants from thethree extractions were combined and 50 μL internal standard(triheptadecanoic acid; 10 mg/mL toluene) was added prior to evaporationto dryness at room temperature under a stream of nitrogen gas; standardscontaining 0, 0.0050, 0.0100, 0.0150, 0.0200 and 0.0300 g soybean oil,in 5 mL heptane, were prepared in the same manner. Fats were convertedto fatty acid methyl esters (FAMEs) by adding 1 mL 5% sulfuric acid(v:v. in anhydrous methanol) to the dried pellets and heating them at80° C. for 30 min, with occasional vortex mixing. The samples wereallowed to cool to room temperature and 1 mL 25% aqueous sodium chloridewas added followed by 0.8 mL heptane. After vortex mixing the phaseswere allowed to separate and the upper organic phase was transferred toa sample vial and subjected to GC analysis.

Plotting NMR determined oil contents versus GC determined oil contentsresulted in a linear relationship between 9.66 and 26.27% oil (GCvalues; % seed wt basis) with a slope of 1.0225 and an R² of 0.9744;based on a seed moisture content that averaged 2.6+/−0.8%.

Seed Oil Content of Arabidopsis Seed was Calculated as Follows:

mg oil=(NMR signal−2.1112)/37.514%

% oil (% wt basis)=(mg oil/1000)/sample weight)*100

Prior to establishing this formula, Arabidopsis seed oil was extractedas follows. Approximately 5 g of mature Arabidopsis seed (cv Columbia)were ground to a fine powder using a mortar and pestle. The powder wasplaced into a 33×94 mm paper thimble (Ahlstrom #7100-3394; Ahlstrom,Mount Holly Springs, Pa., USA) and the oil extracted duringapproximately 40 extraction cycles with petroleum ether (BP 39.9—51.7°C.) in a Soxhlet apparatus. The extract was allowed to cool and thecrude oil was recovered by removing the solvent under vacuum in a rotaryevaporator. Calibration parameters were determined by precisely weighing11 standard samples of partially purified Arabidopsis oil (samplescontained 3.6, 6.3, 7.9, 9.6, 12.8, 16.3, 20.3, 28.2, 32.1, 39.9 and 60mg of partially purified Arabidopsis oil) weighed to a precision of0.0001 g) into 2 mL polypropylene tubes (Corning Inc, Corning N.Y., USA;Part no. 430917) and subjecting them to NMR analysis. A calibrationcurve of oil content (% seed wt basis) to NMR value was established.

Seed for pKR92 YL DGAT2 T2 were grown from a total of 293 independentevents alongside 75 wild type controls. Oil content of YL DGAT2transgenics ranged from 27.9-47.3%. Average oil content was 43.7%. Oilcontent of wild type controls ranged from 39.5-49.6%. Average oilcontent of wt controls was 44.8%.

Seed for pKR92 YL DGAT1/YL DGAT2 T2 were grown from a total of 295independent events alongside 77 wild type controls. Oil content of YLDGAT1/YL DGAT2 transgenics ranged from 34.5-47.6%. Average oil contentwas 44%. Oil content of wild type controls ranged from 41.3-46.6%.Average oil content of wild type controls was 45%. In summary, thesefindings suggest that seed-specific expression of YL DGAT gene does notincrease oil content of arabidopsis seed.

Analysis of the Fatty Acid Profile of Arabdidopsis Seed Expressing YLDGAT2 Alone or in Combination with YL DGAT1:

GC analysis of FAME was employed to investigate if YL DGAT expressionalters the fatty acid profile of arabidopsis seed. Approximately 100 F2seed were dispensed into individual wells of 96 well strip tubes. Fortransesterification, 50 μL of trimethylsulfonium hydroxide (TMSH) and0.5 mL of hexane were added to the each strip tube and incubated for 30min at room temperature while shaking. Fatty acid methyl esters (1 μLinjected from hexane layer) were separated and quantified using aHewlett-Packard 6890 Gas Chromatograph fitted with an Omegawax 320 fusedsilica capillary column (Catalog #24152, Supelco Inc.). The oventemperature was programmed to hold at 220° C. for 2.6 min, increase to240° C. at 20° C./min and then hold for an additional 2.4 min. Carriergas was supplied by a Whatman hydrogen generator. Retention times werecompared to those for methyl esters of standards commercially available(Nu-Chek Prep, Inc.). Results are summarized in TABLE 5.

TABLE 5 avg % range % oleic oleic pKR92 YL DGAT1/YL DGAT2 18.7 15.2-22.3(n = 28) wild type control 15.2 14.5-15.6 (n = 8) pKR92 YL DGAT2 16.414.9-18.4 (n = 26) wild type control 15.7 15.6-16   (n = 7)Results clearly demonstrate that expression of YL DAGT2 and even more soco-expression of YL DGAT1 and YL DGAT2 in Arabidopsis seed leads toincreased incorporation of oleic acid into seed lipids which providesthe first indication that active YL DGAT1 and YL DGAT2 proteins can beproduced in transgenic seed.

Example 5 Expression of Yarrowia lipolytica DGAT Genes in SoybeanSomatic Embryos

TABLE 6 and TABLE 7 list promoter and terminator sequences that wereused in plasmid constructs for seed specific over-expression of YL DGATgenes.

TABLE 6 Seed-specific Promoters Promoter Organism Promoter Referenceβ-conglycinin α′-subunit soybean Beachy et al., EMBO J. 4: 3047-3053(1985) kunitz trypsin inhibitor soybean Jofuku et al., Plant Cell 1:1079-1093 (1989) glycinin Gy1 soybean WO 2004/071467 BD30 (also calledP34) soybean WO 2004/071467

TABLE 7 Transcription Terminators Transcription Terminator OrganismReference phaseolin 3′ bean WO 2004/071467 kunitz trypsin inhibitor 3′soybean WO 2004/071467Construction of a plasmid construct for expression of the YL DGAT1 geneunder control of the glycinin Gy1 promoter (KS349) (FIG. 3A).

The isolation of soybean glycinin Gy1 promoter was performed as follows.Based on the sequences of the soybean glycinin Gy1 gene sequence(GenBank Accession No. X15121; SEQ ID NO:35) in the NCBI database, twooligos with either BamHI or NcoI sites at the 5′ ends were designed toamplify the soybean glycinin Gy1 promoter (SEQ ID NO:36). Theoligonucleotide sequences of these two oligos are as follows:

SEQ ID NO: 37 (oGy1-1): CGCGGATCCTAGCCTAAGTACGTACTCAAAATGCCASEQ ID NO: 38 (oGy1-2): GAATTCCCATGGGGTGATGACTGATGAGTGTTTAAGGAC

Plasmid pKS349 was constructed in many steps from a number of differentintermediate vectors. The amplified soybean glycinin Gy1 promoterfragment was digested with BamHI and NcoI, purified and cloned into theBamHI and NcoI sites of p24K-G4G-@SalI (PCT Application No. WO 98/59062)to give pZBL114 (SEQ ID NO:39). The NcoIl Kpnl fragment containing GUSwas replaced with an NcoIl Kpnl fragment containing a fusion product ofthe soybean GY1 gene (SEQ ID NO:40) and the synthetic barley high lysine8 (BHL8) gene (U.S. Pat. No. 6,800,726 B1) (SEQ ID NO:41;) to makepZBL133 (SEQ ID NO:43). The DNA sequence of the fusion product of soyGY1 gene and BHL8 gene is set forth as SEQ ID NO: 42. The phaseolinterminator was removed from pZBL133 (XbaI/filled in) and replaced withthe phaseolin terminator found in pKS123 (PCT Application No. WO02/08269) (blunt) to give pKS238 (SEQ ID NO:44). The GY1-BHL8 fusion wasreplaced with native GY1 sequence as follows. pKS238 was was digestedwith Kpnl/Bgll, the remaining vector band (5.1 kb) was ligated to a DNAfragment (Bgll/Kpnl) of the native GY1 gene (SEQ ID NO:40) to givepKS240 (SEQ ID NO: 45) The BamHI/SalI fragment containingGy1/GM-GY1/Phas3′ was excised from pKS 240 and ligated to the BamHI/SalIsites of pKS120 (SEQ ID NO:46) to give pKS242 (SEQ ID NO:47). PlasmidpKS120 is identical to pKS123 (supra) with the exception that theHindIII fragment containing Bcon/NotI/Phas3′ cassette was removed. TheNcoI/NotI fragment containing GM-GY1 was replaced with the NcoI/NotIfragment containing YL-DGAT1 from pYDA1 to give pKS349 (SEQ ID NO:48).

Construction of a Construct for Expression of the YL DGAT2 Gene Underthe Control of the Betaconglycinin Promoter (KS362) (FIG. 3B):

Plasmid pKS362 was constructed in many steps from a number of differentintermediate vectors. The AscI cassette containing Kti/NotI/Kti3′ frompKS121 (PCT Application No. WO 02/00904) was blunted into the NotI(filled in) site on pBluescript II SK+(Stratagene) to give pKS121/BS.The NcoI/NotI fragment from pDsRed-Express Vector (Clontech) was bluntedinto the NotI (filled in) site of pKS121/BS to give pDS-RED in KS121/BS(SEQ ID NO:49). The BamHI cassette containing Kti/DsRed/Kti3′ in pDS-REDin KS121/BS (SEQ ID NO:50) was ligated into the BamHI site of pKS123(PCT Application No. WO 02/08269) to give pKS332 (SEQ ID NO:51). Thegene for the YL-DGAT2 was synthesized by PCR with primers to introduceNotI sites at both ends of the gene (see Example 1). The resulting PCRproduct is digested with NotI restriction enzyme and ligated into theNotI site of pKS332 to give pKS362 (SEQ ID NO:52).

Construction of a Control Plasmid (KS352) (FIG. 2A):

Based on the sequences of the cloned soybean P34 promoter (WO2004/071467) (SEQ ID NO:53), two oligos with either BamHI or NotI sitesat the 5′ ends were designed to re-amplify the P34 promoter. Theoligonucleotide sequences of these two oligos are shown as follows:

SEQ ID NO: 54 (oP34-1): CGCGGATCCAACTAAAAAAAGCTCTCAAATTACATTTTGAGSEQ ID NO: 55 (oP34-2): GAATTCGCGGCCGCAACTTGGTGGAAGAATTTTATGATTTGAAAThe re-amplified P34 promoter fragment was digested with BamHI and NotI,purified and cloned into the BamHI and NotI sites of plasmid pZBL115(SEQ ID NO:56) to make pJS89 (SEQ ID NO:57). The pZBL115 plasmidcontains the origin of replication from pRB322, the bacterial HPThygromycin resistance gene driven by T7 promoter and T7 terminator, anda 35S promoter-HPT-Nos3′ gene to serve as a hygromycin resistant plantselection marker. Morteriella alpine delta-6 desaturase gene (U.S. Pat.No. 5,968,809) (SEQ ID NO:58) was cloned into the NotI site of pJS89(SEQID NO:57) in the sense orientation to make the plant expressioncassettes and pJS93 (SEQ ID NO:59).

The P34 promoter was excised from pJS93 (SEQ ID NO:59) using SalI NotIdouble digestion and ligated to SalI/NotI linearized pKS127 vector (U.S.patent application Ser. No. 11/476,510) (SEQ ID NO:60) to give pKS343(SEQ ID NO:61). The BamHI cassette containing Kti/DsRed/Kti3′ in pDS-REDin KS121/BS was blunted and ligated into the HindIII (filled in) site ofpKS343 to give pKS352 (SEQ ID NO:62)

Construction of a Plasmid for Co-Expression of YL DGAT1 and YL DGAT2(KS364):

Plasmid pKS364 (SEQ ID NO:63) was constructed by ligating the 3.3 kbHindIII cassette containing BcongI PRO/YL-DGAT2/Phas TER from pKS362(SEQ ID NO:52) into the unique HindIII site downstream of the of the Gy1promoter in pKS349 (SEQ ID NO:48).

Generation of Transgenic Somatic Embryos:

For co-expression of YL DGAT1 and YL DGAT2 gene in soybean somaticembryos soybean tissue was co-bombarded as described below with amixture of KS349 and KS362. Briefly, DNA of KS349 was digested withrestriction enzymes PstI, XhoI to inactivate the selectable marker genecassette (CaMV35S PRO/HPT/CaMV NOS TER). This DNA was mixed in a 10:1ratio with Sail-linearized plasmid DNA of KS362 and used for soybeantransformation as outlined below. Alternatively, soybean somatic embryossoybean tissue was bombarded as described below with intact plasmid DNAof KS364 which contains functional expression cassettes for both, YLDGAT1 and YL DGAT2. For expression of YL DGAT1 alone, uncut plasmid DNAof KS349 was used for particle bombardment of embryo tissue. Similarly,for expression of YL DGAT2 alone, uncut plasmid DNA of KS362 was usedfor particle bombardment of embryo tissue. Moreover, DNA that containeda selectable marker only (KS352) was used for soybean tissuetransformation in an identical fashion.

Culture Conditions:

Soybean embryogenic suspension cultures (cv. Jack) were maintained in 35mL liquid medium SB196 (infra) on a rotary shaker, 150 rpm, 26° C. withcool white fluorescent lights on 16:8 h day/night photoperiod at lightintensity of 60-85 pE/m2/s. Cultures were subcultured every 7 days totwo weeks by inoculating approximately 35 mg of tissue into 35 mL offresh liquid SB196 (the preferred subculture interval is every 7 days).

Soybean embryogenic suspension cultures were transformed with thesoybean expression plasmids by the method of particle gun bombardment(Klein et al., Nature 327:70 (1987)) using a DuPont Biolistic PDS1000/HEinstrument (helium retrofit) for all transformations.

Soybean Embryogenic Suspension Culture Initiation:

Soybean cultures were initiated twice each month with 5-7 days betweeneach initiation. Pods with immature seeds from available soybean plants45-55 days after planting were picked, removed from their shells andplaced into a sterilized magenta box. The soybean seeds were sterilizedby shaking them for 15 min in a 5% Clorox solution with 1 drop of ivorysoap (i.e., 95 mL of autoclaved distilled water plus 5 mL Clorox and 1drop of soap, mixed well). Seeds were rinsed using 2 1-liter bottles ofsterile distilled water and those less than 4 mm were placed onindividual microscope slides. The small end of the seed was cut and thecotyledons pressed out of the seed coat. Cotyledons were transferred toplates containing SB199 medium (25-30 cotyledons per plate) for 2 weeks,then transferred to SB1 for 2-4 weeks. Plates were wrapped with fibertape. After this time, secondary embryos were cut and placed into SB196liquid media for 7 days.

Preparation of DNA for Bombardment:

Either an intact plasmid or a DNA plasmid fragment containing the genesof interest and the selectable marker gene were used for bombardment.

A 50 μL aliquot of sterile distilled water containing 1 mg of goldparticles was added to 5 μL of a 1 μg/μL DNA solution (either intactplasmid or DNA fragment prepared as described above), 50 μL 2.5M CaCl₂)and 20 μL of 0.1 M spermidine. The mixture was pulsed 5 times on level 4of a vortex shaker and spun for 5 sec in a bench microfuge. After a washwith 150 μL of 100% ethanol, the pellet was suspended by sonication in85 μL of 100% ethanol. Five μL of DNA suspension was dispensed to eachflying disk of the Biolistic PDS1000/HE instrument disk. Each 5 μLaliquot contained approximately 0.058 mg gold particles per bombardment(i.e., per disk).

Tissue Preparation and Bombardment with DNA:

Approximately 100-150 mg of 7 day old embryonic suspension cultures wereplaced in an empty, sterile 60×15 mm petri dish and the dish was placedinside of an empty 150×25 mm Petri dish. Tissue was bombarded 1 shot perplate with membrane rupture pressure set at 650 PSI and the chamber wasevacuated to a vacuum of 27-28 inches of mercury. Tissue was placedapproximately 2.5 inches from the retaining/stopping screen.

Selection of Transformed Embryos:

Transformed embryos were selected using hygromycin as the selectablemarker. Specifically, following bombardment, the tissue was placed intofresh SB196 media and cultured as described above. Six to eight dayspost-bombardment, the SB196 is exchanged with fresh SB196 containing 30mg/L hygromycin. The selection media was refreshed weekly. Four to sixweeks post-selection, green, transformed tissue was observed growingfrom untransformed, necrotic embryogenic clusters. Isolated, greentissue was removed and inoculated into multi-well plates to generatenew, clonally propagated, transformed embryogenic suspension cultures.

Embryo Maturation:

Transformed embryogenic clusters were cultured for one-three weeks at26° C. in SB196 under cool white fluorescent (Phillips cool whiteEconowatt F40/CW/RS/EW) and Agro (Phillips F40 Agro) bulbs (40 watt) ona 16:8 hrphotoperiod with light intensity of 90-120 μE/m² s. After thistime embryo clusters were removed to a solid agar media, SB166, for 1week. Then subcultured to medium SB103 for 3 weeks. Alternatively,embryo clusters were removed to SB228 (SHaM) liquid media, 35 mL in 250mL Erlenmeyer flask, for 2-3 weeks. Tissue cultured in SB228 wasmaintained on a rotary shaker, 130 rpm, 26° C. with cool whitefluorescent lights on 16:8 h day/night photoperiod at light intensity of60-85 pE/m2/s. During this period, individual embryos were removed fromthe clusters and screened for alterations in their fatty acidcompositions as described supra. Media Recipes:

SB 196—FN Lite Liquid Proliferation Medium (Per Liter)

MS FeEDTA -100x Stock 1 10 mL MS Sulfate-100x Stock 2 10 mL FN LiteHalides-100x Stock 3 10 mL FN Lite P, B, Mo-100x Stock 4 10 mL B5vitamins (1 mL/L) 1.0 mL  2,4-D (10 mg/L final concentration) 1.0 mL KNO₃ 2.83 gm (NH₄)₂SO₄ 0.463 gm  Asparagine  1.0 gm Sucrose (1%)   10 gmpH 5.8

FN Lite Stock Solutions

Stock Number 1000 mL 500 mL 1 MS Fe EDTA 100x Stock Na₂ EDTA*  3.724 g 1.862 g FeSO₄—7H₂O  2.784 g  1.392 g 2 MS Sulfate 100x stock MgSO₄—7H₂O 37.0 g   18.5 g MnSO₄—H₂O  1.69 g  0.845 g ZnSO₄—7H₂O  0.86 g   0.43 gCuSO₄—5H₂O 0.0025 g 0.00125 g 3 FN Lite Halides 100x Stock CaCl₂—2H₂O 30.0 g   15.0 g KI  0.083 g  0.0715 g CoCl₂—6H₂O 0.0025 g 0.00125 g 4FN Lite P, B, Mo 100x Stock KH₂PO₄  18.5 g   9.25 g H₃BO₃  0.62 g   0.31g Na₂MoO₄—2H₂O  0.025 g  0.0125 g *Add first, dissolve in dark bottlewhile stirring

SB1 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

31.5 g Glucose

2 mL 2,4-D (20 mg/L final concentration)

pH 5.7

8 g TC agar

SB199 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

30 g Sucrose

4 ml 2,4-D (40 mg/L final concentration)

pH 7.0

2 gm Gelrite

SB 166 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl₂ hexahydrate

5 g Activated charcoal

pH 5.7

2 g Gelrite

SB 103 Solid Medium (Per Liter)

1 package MS salts (Gibco/BRL—Cat. No. 11117-066)

1 mL B5 vitamins 1000× stock

60 g maltose

750 mg MgCl₂ hexahydrate

pH 5.7

2 g Gelrite

SB 71-4 Solid Medium (Per Liter)

1 bottle Gamborg's B5 salts w/sucrose (Gibco/BRL—Cat. No. 21153-036)

pH 5.7

5 g TC agar

2,4-D Stock

Obtain premade from Phytotech Cat. No. D 295—concentration 1 mg/mL

B5 Vitamins Stock (per 100 mL)

Store aliquots at −20° C.

10 g Myo-inositol

100 mg Nicotinic acid

100 mg Pyridoxine HCl

1 g Thiamine

If the solution does not dissolve quickly enough, apply a low level ofheat via the hot stir plate.

SB 228-Soybean Histodifferentiation & Maturation (SHaM) (Per Liter)

DDI H2O 600 ml FN-Lite Macro Salts for SHaM 10X 100 ml MS Micro Salts1000x  1 ml MS FeEDTA 100x  10 ml CaCl 100x 6.82 ml  B5 Vitamins 1000x 1 ml L-Methionine 0.149 g Sucrose   30 g Sorbitol   30 g Adjust volumeto 900 mL pH 5.8 Autoclave Add to cooled media (≤30 C.): *Glutamine(Final conc. 30 mM) 4% 110 mL *Note: Final volume will be 1010 mL afterglutamine addition.

Because glutamine degrades relatively rapidly, it may be preferable toadd immediately prior to using media. Expiration 2 weeks after glutamineis added; base media can be kept longer w/o glutamine.

FN-Lite Macro for SHAM 10×-Stock #1 (Per Liter)

(NH₄)2SO₄ (Ammonium Sulfate) 4.63 g KNO₃ (Potassium Nitrate) 28.3 gMgSO₄*7H₂0 (Magnesium Sulfate Heptahydrate)  3.7 g KH₂PO₄ (PotassiumPhosphate, Monobasic) 1.85 g Bring to volume Autoclave

MS Micro 1000×-Stock #2 (Per 1 Liter)

H₃BO₃ (Boric Acid)   6.2 g MnSO₄*H₂O (Manganese Sulfate Monohydrate) 16.9 g ZnSO4*7H20 (Zinc Sulfate Heptahydrate)   8.6 g Na₂MoO₄*2H20(Sodium Molybdate Dihydrate)  0.25 g CuSO₄*5H₂0 (Copper SulfatePentahydrate)  0.025 g CoCl₂*6H₂0 (Cobalt Chloride Hexahydrate)  0.025 gKI (Potassium Iodide) 0.8300 g Bring to volume Autoclave

FeEDTA 100×-Stock #3 (Per Liter)

Na₂EDTA* (Sodium EDTA) 3.73 g FeSO₄*7H₂0 (Iron Sulfate Heptahydrate)2.78 g Bring to Volume Solution is photosensitive. Bottle(s) should bewrapped in foil to omit light. Autoclave *EDTA must be completelydissolved before adding iron.

Ca 100×-Stock #4 (Per Liter)

CaCl₂*2H₂0 (Calcium Chloride Dihydrate) 44 g Bring to Volume Autoclave

B5 Vitamin 1000×-Stock #5 (Per Liter)

Thiamine*HCl  10 g Nicotinic Acid  1 g Pyridoxine*HCl  1 g Myo-Inositol100 g Bring to Volume Store frozen

4% Glutamine—Stock #6 (Per Liter)

DDI water heated to 30° C. 900 ml L-Glutamine 40 g Gradually add whilestirring and applying low heat. Do not exceed 35° C. Bring to VolumeFilter Sterilize Store frozen * * Note: Warm thawed stock in 31° C. bathto fully dissolve crystals.

Oil Analysis:

Somatic embryos were harvested after two weeks of culture in the liquidmaturation medium SB228 (SHaM) liquid media. Approximately 30 eventswere created in transformations with KS352, KS349/KS362, and KS362 andKS364. All embryos generated for a given event were harvested in bulkand processed as follows. Embryos were frozen on dry ice or byincubation in a −80° C. freezer for two h followed by lyophilization for48 h.

Dried embryos were ground to a fine powder using a genogrinder vial(½″×2″ polycarbonate) and a steel ball (SPEX Centriprep (Metuchen, N.J.,U.S.A.). Grinding time was 30 sec at 1450 oscillations per min. Forevery event, triplicates of approximately 10 mg of tissue were weighedinto Eppendorf tubes. The tissue was extracted using 200 μL heptane atroom temperature under continuous shaking for 2 h. Heptane extracts werecleared by centrifugation and 25 μL of extract was derivatized to fattyacid methyl esters as follows. One mL of a 25% sodium methoxide stocksolution was added to 24 mL of HPLC grade methanol. Sodium methoxide wasstored under an inert gas.

Five μL of a 17:0 TAG (Nu-Chek Prep, Elysian, Minn., USA) stock solution(10 mg/mL) was combined with 25 μL of heptane tissue extract in a glassculture tube 500 μL of 1% sodium methoxide was added. Sample werederivatized in a water bath at 50° C. for 15 min. Samples were allowedto cool to RT and 1 mL of 1M NaCl was added followed by brief mixing.FAMEs were extracted into 1 mL of hepatene and 4 μL sample werequantitated by GC analysis.

Data analysis was performed by plotting the oleic content (% of totalFAME) against the total FAME content (% DW). TABLE 8 shows that somaticembryos generated with a vector control (KS352) show little fluctuationin oleic acid content and some fluctuation in oil content that can verylikely be attributed to biological variation that in introduced in theregeneration process. For example, embryos very likely show variation intheir developmental stage at the time of harvesting. In embryosgenerated with the control construct no correlation (R2=0.1142) wasobserved between the oleic acid content and the oil content (TABLE 8).In embryos generated with plasmid constructs expressing YL DGAT1 and YLDGAT2 s gene alone, KS349 and KS362, respectively or both YL DGAT1 andDGAT2 genes (KS349/KS362, KS364) under control of strong seed specificpromoters both oleic acid content and total esterified fatty acidcontent showed a wide range of fluctuation. Moreover, as shown in FIGS.4B and 5B, a strong correlation (R²≥0.59) was observed between the oleicacid content and the total esterified fatty acid content for somaticembryos generated with KS349 and KS362 either alone or in combinationsas well as with KS364, a transformation plasmid that contains expressioncassettes for YL DGAT1 and YL DAT2 genes.

TABLE 8 Esterified Fatty Acid and Oleic Acid Content of Soybean SomaticEmbryos oleic oleic FAME acid FAME acid Event (% (% total Event (% (%total # DCW) stdv FAME) # DCW) stdv FAME) KS352 KS352 22 6.2 nd 18.9 444.6 nd 17.1 16 5.7 nd 15.8 65 4.6 nd 19.4 35 5.6 nd 19.5 6 4.5 nd 13.948 5.6 nd 18.5 24 4.5 nd 16.1 14 5.5 nd 17.3 31 4.5 nd 15.9 43 5.5 nd18.7 20 4.4 nd 17.1 42 5.4 nd 19.3 37 4.4 nd 17.3 33 5.3 nd 17.2 69 4.4nd 19.2 68 5.3 nd 18.6 50 4.3 nd 17.2 3 5.2 nd 18.5 54 4.3 nd 19.5 4 5.2nd 18.9 55 4.3 nd 16.1 11 5.2 nd 19.1 64 4.3 nd 18.7 41 5.2 nd 16.9 324.1 nd 14.4 51 5.2 nd 18.2 61 4.1 nd 16.8 7 5.1 nd 17.2 23 4.0 nd 16.110 5.1 nd 19.9 26 4.0 nd 13.6 21 5.1 nd 18.2 49 4.0 nd 16.5 27 5.1 nd18.3 18 3.9 nd 16.4 1 5.0 nd 17.6 8 3.8 nd 15.5 46 5.0 nd 18.4 53 3.8 nd20.2 59 5.0 nd 17.5 63 3.8 nd 17.2 66 5.0 nd 19.3 52 3.7 nd 17.3 5 4.9nd 15.1 17 3.6 nd 14.3 15 4.9 nd 15.7 36 3.6 nd 15.7 29 4.9 nd 16.5 603.4 nd 16.6 2 4.8 nd 17.6 12 3.3 nd 15.4 9 4.8 nd 17.4 45 3.3 nd 15.2 304.8 nd 17.0 62 3.3 nd 18.8 34 4.8 nd 16.9 40 3.2 nd 13.3 19 4.7 nd 14.825 3.0 nd 12.3 47 4.7 nd 17.4 38 3.0 nd 16.2 67 4.7 nd 22.9 57 2.5 nd18.0 13 4.6 nd 17.2 56 2.3 nd 18.2 28 4.6 nd 15.9 58 2.2 nd 16.5 39 4.6nd 18.6 KS349 KS362 28 10.8 1.0 33.2 28 14.7 0.3 28.9 16 10.6 3.1 32.716 14.5 0.1 33.1 25 10.3 0.9 34.1 19 14.1 0.6 32.6 19 9.7 0.2 31.5 2413.6 0.0 29.9 8 9.5 0.9 34.7 17 13.3 0.4 31.3 7 9.4 0.3 34.3 21 12.7 0.228.0 18 9.3 0.1 34.3 12 12.6 0.1 30.6 30 9.2 0.0 32.3 6 11.6 1.3 32.0 38.6 0.6 32.9 18 11.5 0.6 26.5 20 8.5 0.1 32.9 22 10.9 0.7 23.8 24 8.40.1 30.8 11 10.8 0.1 27.2 5 8.3 0.3 37.0 26 10.8 0.1 25.8 21 8.2 0.532.5 14 10.6 0.2 25.3 12 8.1 0.3 33.2 13 10.4 0.3 22.4 31 8.1 0.2 33.420 9.8 0.3 21.1 11 7.5 0.5 30.5 8 9.5 0.7 21.8 6 7.5 0.2 29.9 25 9.3 0.119.5 22 7.5 0.7 33.2 10 9.1 0.5 21.1 27 7.1 0.4 19.4 4 8.8 0.3 23.5 297.1 1.5 29.9 15 8.8 0.0 19.1 23 6.6 0.3 18.4 23 7.8 0.2 16.1 17 6.3 0.722.7 27 7.8 0.6 18.3 15 6.0 0.1 28.1 1 7.2 0.6 20.1 4 5.7 0.1 21.2 3 7.20.3 18.1 13 5.7 0.0 23.6 9 7.2 0.2 14.8 14 5.6 0.2 21.7 2 6.3 0.3 17.526 5.5 0.1 19.1 7 6.1 0.3 22.9 10 5.5 0.1 30.8 5 4.6 0.2 17.3 2 5.5 0.328.2 9 5.2 0.2 16.6 1 4.6 0.3 17.1 KS349/KS362 KS364 12 13.2 0.3 34.1 2116.1 0.9 35.9 4 11.7 0.7 33.4 29 14.6 2.1 33.8 13 11.4 0.2 33.1 18 14.22.3 33.2 18 11.1 0.2 33.5 27 13.4 0.8 31.7 24 11.1 0.1 33.3 20 12.2 0.835.6 3 10.9 0.0 34.1 28 11.8 0.7 34.6 10 10.8 0.2 31.8 26 11.5 1.6 30.89 10.7 0.2 33.5 3 11.3 1.0 36.2 23 10.6 0.4 32.7 22 10.9 1.4 34.0 1710.2 0.8 31.5 1 10.7 0.6 30.8 29 10.0 0.5 26.6 24 10.7 0.6 31.2 11 9.90.3 31.3 25 10.6 0.2 31.2 16 9.4 0.1 31.7 6 10.2 0.4 32.6 19 9.3 0.328.1 2 10.1 0.3 31.0 1 8.9 0.5 27.4 5 9.9 0.5 33.3 25 8.5 0.5 31.8 119.8 0.4 37.8 7 8.4 0.0 17.6 12 9.8 0.6 37.0 15 8.4 0.1 29.8 8 9.4 0.131.5 26 8.3 0.2 18.6 10 9.3 0.1 31.2 6 8.1 0.2 24.5 15 9.2 0.4 33.7 57.4 0.1 19.3 16 9.1 0.2 33.9 21 7.4 0.8 18.0 19 8.7 0.1 27.3 27 6.9 0.825.7 7 8.4 0.6 26.7 30 6.9 0.7 24.7 4 7.6 0.1 28.1 2 6.0 0.3 18.2 23 5.70.2 21.1 14 5.6 0.4 23.0 9 4.2 0.3 15.1 22 5.6 0.2 17.6 14 4.2 0.2 15.18 4.6 0.1 17.4 17 3.6 0.4 21.9 28 4.2 0.2 19.7 13 3.5 0.1 15.0 20 2.80.1 11.9

In summary, the data shows that in soybean somatic embryos, similar toArabidopsis seed YL DGAT gene expression is associated with increasedincorporation of oleic acid into the total esterified fatty acidfraction. However, in contrast to Arabidopsis, in soybean somaticembryos increased oleic acid content is tightly correlated with totalaccumulation of esterified fatty acid. In other words, expression of YLDGAT2 alone and co-expression of YL DGAT1 and YL DAGT2 in soybeansomatic embryos leads to increased biosynthesis and incorporation offatty acids into the total esterified fatty acid fraction. Takentogether this finding strongly suggests that expression of YL DGAT genesprovides an efficient strategy to achieve an increase in the total ofoil content of soybean seed.

Example 6

Expression of Yarrowia lipolytica DGAT Genes in Soybean Seed

Construction of a control plasmid (KS332) (FIG. 2B) containing only CaMV35S PRO/HPT/NOS TER and Kti PRO/DsRed/Kti TER expression cassettes isdescribed in Example 5. Its sequence is set forth as SEQ ID NO:51.

Transgenic soybean lines were generated by the method of particle gunbombardment (Klein et al., Nature (London) 327:70-73 (1987); U.S. Pat.No. 4,945,050) using a BIORAD Biolistic PDS1000/He instrument andplasmid DNA of KS332, KS362 and a 10:1 mixture of KS349 and KS362prepared as described in Example 3. The following stock solutions andmedia were used for transformation and regeneration of soybean plants:

Stock Solutions: Sulfate 100× Stock:

37.0 g MgSO₄.7H₂O, 1.69 g MnSO₄.H₂O, 0.86 g ZnSO₄.7H₂O, 0.0025 g

CuSO₄.5H₂O

Halides 100× Stock:

30.0 g CaCl₂.2H₂O, 0.083 g KI, 0.0025 g CoCl₂.6H₂O P, B, Mo 100× Stock:

18.5 g KH₂PO₄, 0.62 g H₃BO₃, 0.025 g Na₂MoO₄.2H₂O Fe EDTA 100× Stock:

3.724 g Na₂EDTA, 2.784 g FeSO₄.7H₂O

2,4-D Stock:

10 mg/mL Vitamin B5 1000× Stock: 10.0 g myo-inositol, 0.10 g nicotinicacid, 0.10 g pyridoxine HCl, 1 g thiamine.

Media (per Liter):

SB196: 10 mL of each of the above stock solutions, 1 mL B5 Vitaminstock,

0.463 g (NH₄)₂SO₄, 2.83 g KNO₃, 1 mL 2,4-D stock, 1 g asparagine, 10 gSucrose, pH 5.7

SB103:

1 pk. Murashige & Skoog salts mixture, 1 mL B5 Vitamin stock, 750 mgMgCl₂

hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.

SB166:

SB103 supplemented with 5 g per liter activated charcoal.

SB71-4:

Gamborg's B5 salts, 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH5.7.

To prepare tissue for transformation, soybean embryogenic suspensioncultures were maintained in 35 mL liquid medium (SB196) on a rotaryshaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hday/8 h night cycle. Cultures were subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of fresh liquidmedia.

In particle gun bombardment procedures it is possible to use purified 1)entire plasmid DNA; or 2) DNA fragments containing only the recombinantDNA expression cassette(s) of interest. For every seventeen bombardmenttransformations, 85 μL of suspension is prepared containing 1 to 90picograms (pg) of plasmid DNA per base pair of each DNA plasmid. Bothrecombinant DNA plasmids were co-precipitated onto gold particles asfollows. The DNAs in suspension were added to 50 μL of a 20-60 mg/mL 0.6μm gold particle suspension and then combined with 50 μL CaCl₂ (2.5 M)and 20 μL spermidine (0.1 M). The mixture was vortexed for 5 sec, spunin a microfuge for 5 sec, and the supernatant removed. The DNA-coatedparticles were then washed once with 150 μL of 100% ethanol, vortexedand spun in a microfuge again, then resuspended in 85 μL of anhydrousethanol. Five μL of the DNA-coated gold particles were then loaded oneach macrocarrier disk.

Approximately 150 to 250 mg of two-week-old suspension culture wasplaced in an empty 60 mm×15 mm petri plate and the residual liquidremoved from the tissue using a pipette. The tissue was placed about 3.5inches away from the retaining screen and each plate of tissue wasbombarded once. Membrane rupture pressure was set at 650 psi and thechamber was evacuated to −28 inches of Hg. Three plates were bombarded,and, following bombardment, the tissue from each plate was dividedbetween two flasks, placed back into liquid media, and cultured asdescribed above.

Seven days after bombardment, the liquid medium was exchanged with freshSB196 medium supplemented with 30-50 mg/L hygromycin. The selectivemedium was subsequently refreshed weekly or biweekly. Seven weekspost-bombardment, bright green, transformed tissue was observed growingfrom untransformed, chlorotic or necrotic embryogenic clusters. Isolatedgreen tissue was removed and inoculated into individual wells insix-well culture dishes to generate new, clonally-propagated,transformed embryogenic suspension cultures. Thus, each new line wastreated as independent transformation event in an individual well. Thesesuspensions can then be maintained as suspensions of embryos clusteredin an immature developmental stage through subculture or they can beregenerated into whole plants by maturation and germination ofindividual somatic embryos.

After two weeks in individual cell wells, transformed embryogenicclusters were removed from liquid culture and placed on solidifiedmedium (SB166) containing no hormones or antibiotics for one week.Embryos were cultured for at 26° C. with mixed fluorescent andincandescent lights on a 16 h day/8 h night schedule. After one week,the cultures were then transferred to SB103 medium and maintained in thesame growth conditions for 3 additional weeks.

Somatic embryos became suitable for germination after four weeks andwere then removed from the maturation medium and dried in empty petridishes for one to five days. The dried embryos were then planted inSB71-4 medium where they were allowed to germinate under the same lightand temperature conditions as described above. Germinated embryos weretransferred to sterile soil and grown to maturity for seed production.

A total of 29 transgenic lines with seed were generated with intactplasmid DNA of KS362 at concentration of 15 μg per bp of plasmid DNA pergold particle preparation (see above). For every event 20 seed werescored for the presence of the DS marker gene. Briefly, seeds wereobserved under a stereo microscope (Leica MZ Fluo III) using a UV lightsource. A filter set customized for fluorescence associated with DsRedexpression with the follwing properties was used: Exitation λ=540-580nm/Emssion λ≥570 nm. In cases were less that 20 seed were available allseed were scored in this manner. Subsequently soybean seed oil contentwas measured by NMR as described previously 3. Nineteen events generatedwith KS362 contained seed that were positive for DsRed. Of these, 11events showed a detectable difference in oil content between DsRedpositive transgenic segregants and DsRed negative null segregants. Dataare summarized in TABLE 9.

TABLE 9 Oil Content of T1 Soybean Seed Generated with KS362 avg % avg %delta delta n oil null n oil DsRed+ % points % AFS4822.4.5.1 5 13.3 1520.4 7.2 54.1 AFS4822.3.2.1 10 14.2 9 18.9 4.7 32.7 AFS4822.3.3.1 5 12.48 16.1 3.7 29.5 AFS4822.4.2.1 4 16.2 6 20.9 4.7 29.3 AFS4822.4.1.1 915.1 8 19.4 4.3 28.9 AFS4822.1.13.1 5 15.3 15 19.1 3.7 24.4AFS4822.1.2.1 2 15.8 18 19.6 3.8 24.1 AFS4822.4.17.1 7 14.9 13 18.5 3.623.9 AFS4822.1.9.1 8 16.9 12 19.8 3.0 17.5 AFS4822.2.11.1 6 17.9 14 20.72.8 15.7 AFS4822.2.10.1 6 20.6 14 23.5 2.8 13.7

A total of 10 transgenic lines with seed were generated with DNA ofKS332. For every event 20 seed were scored for the presence of the DSmarker gene as described above. In cases were less that 20 seed wereavailable all seed were scored in this manner. Subsequently soybean seedoil content was measured by NMR as described in Example 4. Seven eventsgenerated with KS 332 contained seed that were positive for DsRed. Dataare summarized in TABLE 10.

TABLE 10 Oil Content of T1 Soybean Seed Generated with KS332 avg % avg %delta delta n oil null n oil DsRed+ % points % AFS4703.1.1.1 3 21.8 1720.5 −1.4 −6.2 AFS4703.1.2.1 4 20.8 16 18.5 −2.3 −11.2 AFS4703.1.6.1 520.4 15 21.4 1.0 5.0 AFS4703.2.3.1 8 22.1 12 21.1 −0.9 −4.1AFS4703.2.4.1 6 22.2 14 21.7 −0.4 −2.0 AFS4703.3.8.1 6 21.8 14 21.8 0.00.2 AFS4703.3.16.1 5 23.9 15 23.3 −0.6 −2.5

In contrast to seed generated with KS362, for transgenic seed generatedwith KS332 no consistent oil increase could be associated with thepresence of the DsRed marker in T1 segregants.

Four events generated with KS362 were subjected to analysis of DsRedstatus and oil NMR of all available T1 seed. Data are summarized inTABLE 11.

TABLE 11 Oil Content of T1 Soybean Seed Generated with KS362 avg % avg %delta delta n oil null n oil DS red+ % points % AFS4822.4.5.1 8 12.0 2418.3 6.3 52.9 AFS4822.1.13.1 17 15.0 42 18.7 3.7 24.6 AFS4822.1.2.1 415.7 40 19.3 3.5 22.3 AFS4822.2.10.1 8 19.5 25 23.0 3.5 17.8AFS4822.2.11.1 6 17.9 17 20.3 2.4 13.5

In summary, the data in the previous tables and FIGS. 7A and 7B showthat seed specific expression of YL DAGT2 leads to increased oilbiosynthesis during soybean seed maturation and thus provides anefficient metabolic engineering tool to increase oil accumulation insoybeans.

A total of 16 transgenic lines with seed were generated byco-bombardment with DNA of KS349 and KS362 that had been mixed at a 10:1ratio (see Example 4). The DNA mixture was delivered in soybeantransformation at a final concentration of 15 μg per bp of plasmid DNAper gold particle preparation. Briefly, prior to bombardment DNA KS349was digested with PstI, XhoI for inactivation of the selectable markergene on this plasmid. DNA of KS362 was linearized with SalI.

It is reasonable to assume that because of the pre-treament of the DNA,the selectable marker gene in all transformations was delivered from theplasmid (KS362) that was bombarded at the lower DNA concentration.Initial inspection of these seed under the fluorescencestereo-microcsope revealed that very few events of this transformationcontained T1 seed that were positive for the DsRed marker gene. Thisresult may be due to the proximity of the DsRed expression cassette tothe end of the SalI restriction fragment of KS362 that was used forsoybean transformation. It may have resulted in the integration of KS362DNA fragments that did not contain a functional DsRed expressioncassette. For this reason T1 seed were screened for the absence ofpresence of the transgene-derived YL DGAT by assaying the seed fattyacid composition. For every event 20 seed were analyzed by GC. 50 seedof untransformed soybean seed were processed in the same manner. Soybeanseed chips were produced by cutting the seed with a razorblade avoidingthe embryonic axis. Seed chips of approximately 2 mg were placed in avial containing 50 μL trimethylsulfonium hydroxide and 0.5 mL hexane.The chips were incubated for 30 min at room temperature while shaking. 5μL of the hexane layer was injected into a Hewlett Packard 6890 GasChromatograph containing a Omegawax 320 fused silica capillary column(Supelco Cat. No. 24152). Oven conditions were as follows: initialtemperature of 220° C. for 2.7 minutes, ramped to 240° C. over 1 min andheld at 240° C. For a total run time of 6 min. Retention times werecompared to standards commercially available (Nu-Chek Prep, Inc. Cat.No. U-99-A). Fatty acids were determined by direct trans-esterificationof individual standards in 0.5 mL of methanolic H₂SO₄ (2.5%). Fatty acidmethyl esters were extracted from the methanolic solutions into hexaneafter the addition of an equal volume of water.

Ten events were identified that contained T1 seed with 25% oleic acidcontent. Since this oleic acid content was not observed in untransformedsoybean seed (see FIG. 6A and TABLE 12) and increased oleic acid contentwas previously associated with YL DGAT2 and YL DGAT1 and YL DGAT2co-expression both in arabidopsis seed and soybean somatic embryos, itis believed that the presence of oleic acid at levels of 25% providesefficient means to identify YL DGAT positive T1 seed. After GC analysisfor YL DGAT genotyping, seed were subjected to oil measurements by NMRas described previously (Example 3). When oleic acid content was plottedagainst total oil content seven of the 10 events with T1 seed of 25%showed a correlation of R²≥0.3 between oil and oleic acid content. Theproperties of these events are described in more detail in TABLE 12.

TABLE 12 Oil Content of T1 Soybean Seed Generated with KS349/KS362 avg %avg % oil oil delta R² % <25% ≥25% % delta oleic/% n oleic n oleicpoints % oil AFS4818.3.1.1 15 11.9 5 16.8 4.9 41.0 0.38 AFS4818.1.5.1 1015.5 10 20.0 4.5 28.7 0.69 AFS4818.2.10.1 10 13.2 10 16.5 3.3 25.2 0.39AFS4818.1.9.1 5 12.9 14 15.9 3.0 22.9 0.51 AFS4818.2.6.1 12 18.6 8 21.93.4 18.1 0.51 AFS4818.1.3.1 6 20.5 14 23.6 3.1 14.9 0.45 AFS4818.1.2.1 619.4 14 21.8 2.4 12.4 0.37 Jack 56 21.7 0.003 (wt control)Jack wild type seed were grown under similar condition to those used forT1 seed generation and analyzed by GC and NMR analysis. It was observedthat oleic acid and oil content fluctuated between 7.6-20.5% and18.6-25.2%, respectively. No correlation between oleic acid content andoil content could be observed in untransformed soybean seed.

Four events generated with KS349/KS362 were subjected to GC and NMRanalysis of all available T1 seed. Data are summarized in TABLE 13.

TABLE 13 Oil Content of T1 Soybean Seed Generated with KS349/KS362 avg %avg % oil oil delta R² % <25% ≥25% % delta oleic/% n oleic n oleicpoints % oil AFS4818.1.5.1 21 16.3 21 20.2 3.9 23.8 0.55 AFS4818.2.6.149 18.1 23 21.9 3.7 20.7 0.50 AFS4818.1.3.1 33 20.2 67 23.5 3.3 16.20.43 AFS4818.1.2.1 13 19.3 26 22.1 2.7 14.1 0.45Taken together the data in the previous tables and FIGS. 6B and 7Astrongly support the conclusion that co-expression of YL DGAT1 and YLDGAT2 genes, like expression of the YL DGAT2 gene alone, provides anefficient strategy to achieve an increase in the total of oil content ofsoybean seed. Additionally, it should be noted that a high number ofevents could be identified with an oil difference of 2% points betweennull and transgenic segregants among a small set of transgenic eventsscreened.

Example 7 Expression of Yarrowia lipolytica DGAT Genes in Maize

Based on results disclosed in Examples 4, 5 and 6 of the instantapplication, the YL DGAT1 and YL DGAT2 genes can be expressed in theseed embryo of maize to increase the oil content of this tissue. Asdescribed below, this result can be achieved by transforming maize withexpression cassettes comprising open reading frames of DGAT1 and DGAT2from Yarrowia lipolytica operably linked on their 5′ ends to embryopreferred promoters, such as the promoter for the maize 16 kDa oleosingene (Lee, K. and Huang, A. H., Plant Mol. Biol. 26:1981-1987 (1984))and maize embryo abundant (EAP1) promoter and terminator (US 2006272058A1).

An expression cassette comprising the promoter from the maize 16 kDaoleosin gene (OLE PRO), the coding sequence of the YL DGAT2 gene (SEQ IDNO:9) and the polyadenylation signal sequence/terminator from thenopaline synthase (NOS) gene of Agrobacterium tumefaciens is constructedusing methods and technologies known in the art. A second expressioncassette comprises the YL DGAT1 gene under the transcriptional controlof the maize embryo abundant protein (EAP1) promoter and terminator,with the maize ADH1 INTRON1 inserted between the promoter and codingsequence for enhanced expression. The two expression cassettes arelinked, together with a gene encoding a selectable marker, in a binaryvector suitable for Agrobacterium-mediated transformation of maize.

An Agrobacterium-based protocol can be used for the transformation ofmaize (see below). The resulting binary vector is introduced intoAgrobacterium LBA4404 (PHP10523) cells, preferably by electroporation.An in vivo recombination generates a cointegrate plasmid between theintroduced binary vector and the vir plasmid (PHP10523) resident in theAgrobacterium cells. The resulting Agrobacterium cells are used totransform maize.

Transformation of Maize Mediated by Agrobacterium:

Freshly isolated immature embryos of maize, about ten days afterpollination (DAP), can be incubated with the Agrobacterium. Thepreferred genotype for transformation is the highly transformablegenotype Hi-II (Armstrong, Maize Gen. Coop. Newsletter 65:92-93 (1991)).An F1 hybrid created by crossing a Hi-II with an elite inbred may alsobe used. After Agrobacterium treatment of immature embryos, the embryoscan be cultured on medium containing toxic levels of herbicide. Onlythose cells that receive the herbicide resistance gene, and the linkedgene(s), grow on selective medium. Transgenic events so selected can bepropagated and regenerated to whole plants, produce seed, and transmittransgenes to progeny.

Preparation of Agrobacterium:

The engineered Agrobacterium tumefaciens LBA4404 can be constructed tocontain plasmids for seed-preferred expression of YL DGAT1 and YL DGAT2genes, as disclosed in U.S. Pat. No. 5,591,616 (the contents of whichare hereby incorporated by reference). To use the engineered constructin plant transformation, a master plate of a single bacterial colonytransformed with plasmids for seed-preferred expression of YL DGAT1 andYL DGAT2 genes can be prepared by inoculating the bacteria on minimal ABmedium and allowing incubation at 28° C. for approximately three days.(The composition and preparation of minimal AB medium has beenpreviously described in PCT Publication No. WO 02/009040 (the contentsof which are hereby incorporated by reference). A working plate can thenbe prepared by streaking the transformed Agrobacterium on YP medium(0.5% (w/v) yeast extract, 1% (w/v) peptone, 0.5% (w/v) sodium chloride,1.5% (w/v) agar) that contains 50 μg/mL of spectinomycin.

The transformed Agrobacterium for plant transfection and co-cultivationcan then be prepared one day prior to maize transformation. Into 30 mLof minimal A medium (prepared as described in PCT Publication No. WO02/009040) in a flask was placed 50 μg/mL spectinomycin, 100 μMacetosyringone, and about a ⅛ loopful of Agrobacterium from a one totwo-day-old working plate. The Agrobacterium can then be grown at 28° C.with shaking at 200 rpm for approximately fourteen h. At mid-log phase,the Agrobacterium can be harvested and resuspended at a density of 3 to5×108 CFU/mL in 561Q medium that contains100 μM acetosyringone usingstandard microbial techniques. The composition and preparation of 561Qmedium was described in PCT Publication No. WO 02/009040.

Immature Embryo Preparation:

Nine to ten days after controlled pollination of a maize plant,developing immature embryos are opaque and 1-1.5 mm long. This length isthe optimal size for infection with the PHP18749-transformedAgrobacterium. The husked ears can be sterilized in 50% commercialbleach and one drop Tween-20 for thirty minutes, and then rinsed twicewith sterile water. The immature embryos can then be aseptically removedfrom the caryopsis and placed into 2 mL of sterile holding solutionconsisting of medium 561Q that contains 100 μM of acetosyringone.

Agrobacterium Infection and Co-Cultivation of Embryos:

The holding solution can be decanted from the excised immature embryosand replaced with transformed Agrobacterium. Following gentle mixing andincubation for about five minutes, the Agrobacterium can be decantedfrom the immature embryos. Immature embryos were then moved to a plateof 562P medium, the composition of which has been previously describedin PCT Publication No. WO 02/009040. The immature embryos can be placedon this media scutellum surface pointed upwards and then incubated at20° C. for three days in darkness. This step can be followed byincubation at 28° C. for three days in darkness on medium 562P thatcontains 100 μg/mL carbenecillin as described in U.S. Pat. No.5,981,840.

Selection of Transgenic Events:

Following incubation, the immature embryos can be transferred to 5630medium, which can be prepared as described in PCT Publication No. WO02/009040. This medium contains Bialaphos for selection of transgenicplant cells as conferred by the BAR gene that is linked to barley HGGTexpression cassette. At ten to fourteen-day intervals, embryos weretransferred to 5630 medium. Actively growing putative transgenicembryogenic tissue can be after six to eight weeks of incubation on the5630 medium.

Regeneration of T₀ Plants:

Transgenic embryogenic tissue is transferred to 288 W medium andincubated at 28° C. in darkness until somatic embryos matured, or aboutten to eighteen days. Individual matured somatic embryos withwell-defined scutellum and coleoptile are transferred to 272 embryogermination medium and incubated at 28° C. in the light. After shootsand roots emerge, individual plants are potted in soil and hardened-offusing typical horticultural methods.

288 W medium contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS Salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSVitamins Stock Solution (Gibco); 1 mL of zeatin (5 mg/mL solution); 60 gsucrose; 8 g of agar (Sigma A-7049, Purified), 2 mL of indole aceticacid (0.5 mg/mL solution*); 1 mL of 0.1 mM ABA*; 3 mL of Bialaphos (1mg/mL solution*); and 2 mL of carbenicillin (50 mg/mL solution). The pHof this solution is adjusted to pH 5.6. The solution is autoclaved andingredients marked with an asterisk (*) are added after the media hascooled to 60° C.

Medium 272 contains the following ingredients: 950 mL of deionizedwater; 4.3 g of MS salts (Gibco); 0.1 g of myo-inositol; 5 mL of MSvitamins stock solution (Gibco); 40 g of Sucrose; and 1.5 g of Gelrite.This solution is adjusted to pH 5.6 and then autoclaved.

Example 8 Analysis of Kernel Oil Content Nuclear Magnetic Resonance(NMR) Analysis:

Seed are imbibed in distilled water for 12-24 hours at 4° C. The embryois dissected away and stored in a 48 well plate. The samples arelyophilized over-night in a Virtis 24×48 lyophilizer. The NMR (ProcessControl Technologies—PCT (Ft. Collins, Colo.) is set up as per themanufacturer's instructions. The NMR is calibrated using a series of 5mm NMR tubes containing precisely measured amounts of corn oil (Mazola).The calibration standards are 3, 6, 9, 12, 15, 18, 21, 27, 33, and 40 mgof oil.

Example 9 Synthesis of YL DGAT1 and YL DGAT2 genes

Nucleotide sequences encoding YL DGAT1 and YL DGAT2 were designed foroptimized expression in soybean seed using methods similar to thosedescribed in Wu, G et al. Nucleic Acids Research (2007), 35: D76-D79;Villalobos, A. et al. BMC Bioinformatics (2006), 7 No pp. given; Wu, G.et al. Protein Expression and Purification (2006), 47: 441-445;Richardson, S. M. et al. Genome Research (2006), 16: 550-556; Jayaraj,S. et al. Nucleic Acids Research (2005) 33: 3011-3016. DNA moleculeswere synthezised by DNA 2.0 (Menlo Park, Calif., USA).Expression-optimized DNA sequences of YL DGAT1 and YL DGAT2 are setforth in SEQ ID NO:64 and SEQ ID NO:66, respectively. The amino acidsequences for soy optimized enzymes are set forth in SEQ ID NO:65 (YLDGAT1) and SEQ ID NO:67 (YL DGAT2) and are identical to the translationproducts of SEQ ID NO:1 and SEQ ID NO:9, respectively.

Example 10 Fatty Acid Composition of Soybean Somatic Embryos ExpressingYL DGAT Genes

Transgenic somatic embryos were generated using the plasmid constructsKS352, KS349, KS362 and KS364. Generation of the DNA constructs and thetransformation process is described in detail in EXAMPLE 5. Fatty acidcomposition was determined by GC analysis of fatty acid methyl estersgenerated by sodium methoxide derivatization of heptane extracts. Thefindings are summarized in TABLE 14. The table compares the fatty acidcomposition of 100 events generated with a control plasmid lacking YLDGAT genes with that of events created with plasmids containing YL DGAT1(KS349), YL DGAT2 (KS362) or both genes (KS364). For events generatedwith YL DGAT containing DNA constructs the average fatty acidcomposition of all events with greater than 30% oleic is shown.

TABLE 14 Fatty Acid Composition of Soybean Somatic Embryos generatedwith KS 352, 349, 362, 349&362 and 364 palmitic stearic oleic linoleiclinolenic Plasmid n acid acid acid acid acid KS352 100 15.9 5.2 17.944.1 16.9 average KS352 range 12.6-20.8 4.2-6.6 12.3-22.9 39.3-46.912.4-23.5 KS349 18 11.6 5.4 33.0 41.3 8.6 average (>30% oleic) KS349range 10.7-12.8 4.2-6.5 30.5-37.0 38.7-44.6  7.8-10.7 (>30% oleic) KS3625 11.5 6.3 31.9 43.2 6.9 average (>30% oleic) KS362 range 10.9-12.75.7-7.0 30.6-33.1 41.9-44.8  6.2-7.7 (>30% oleic) KS349&362 14 12.8 5.433.8 39.7 8.4 average (>30% oleic) KS349&362 11.5-14.4 3.8-7.0 30.8-35.5  38-42.7  6.3-10.3 range (>30% oleic) KS364 14 10.9 6.4 39.0 38 5.7average (>30% oleic) KS364 range  9.2-12.6 5.9-7.7 32.8-48.6 31.4-42.7 3.2-6.5 (>30% oleic)

The table shows that expression of YL DGAT1 or YL DGAT2 as well asco-expression of said genes alters the FA profile of soybean somaticembryos. The most pronounced alteration is an increase in oleic acid anda decrease in linolenic acid that is consistently observed with all DNAconstructs tested. Expression of YL DGAT genes also leads to a decreasein palmitic and linoleic acid and an increase in stearic acid.

Example 11 Fatty Acid Composition of Soybean Seed Expressing YL DGATGenes

Event AFS4822.1.13.1 was generated using plasmid DNA of KS362 asdescribed in EXAMPLE 6. Transgenic T1 seed show an increase in oilcontent of 24.6% when compared to null segregant seed from the same T1plant. This observation strongly supports the conclusion that thisevents expresses YL DGAT2. T1 seed of AFS4822.1.13.1 with or without theYL DGAT2 transgene were germinated and grown in the growth chamber forthree month. DS-red positive T1 seed of event AFS4703.1.6 weregerminated and grown alongside the YL DGAT2 event. AFS4703.1.6 wasgenerated with KS332, a plasmid vector hat contains the DS-red markergene but does not contain YL DGAT2 (see EXAMPLE 5). Several T1segregants could be identified that only produced DS-red positive T2seed indicating that these lines were homozygous for the respectivetransgene. T2 seed harvested from null-segregant progeny were DS-rednegative confirming that these lines likely did not contain a functionaltransgene.

For each selection six seed were chipped and the fatty acid compositionof the seed chips was analyzed by TMSH-derivatization followed by gaschromatography as described in EXAMPLE 6. TABLE 15A compares the averagefatty acid composition of six seed chips of DS-red positive segregantsof AFS4822.1.13.1 with that of seed chips derived form a null-segregantplant and that of seed chips of event AFS4703.1.6 containing only theDS-red marker gene. It demonstrates that expression of YL DGAT2 altersthe FA profile of soybean seed. The most pronounced alteration is anincrease in oleic acid and a decrease in linolenic acid. Expression ofYL DGAT genes also leads to a decrease in palmitic and linoleic acid andan increase in stearic acid.

TABLE 15A Fatty Acid Composition of T2 Soybean Seed Generated with KS362palmitic stearic oleic linoleic linolenic Event n acid acid acid acidacid AFS4822.1.13.1 6 10.8 4.6 29.2 49.5 6.0 average (DS red positive)AFS4822.1.13.1 6 12.0 3.4 16.3 59.0 9.4 average (Null segregant)AFS4703.1.6 6 11.5 3.3 15.8 59.8 9.6 average (DS red positive)

Events AFS4818.1.9 and AFS4818.1.3 were generated using a mixture of DNAfragments derived from plasmids KS349 (YL DGAT1) and KS362 (YL DGAT2) asdescribed in EXAMPLE 5. Transgenic T1 seed of these two events show anincrease in oil content of 22.9 and 16.2%, respectively when compared tonull segregant seed from the same T1 plant. Although this observationstrongly supports the conclusion that both events express transgenederived YL DGAT it is not clear if both events contain intact copies ofboth or just one DGAT gene present in the DNA mixture used fortransformation. T1 seed with increased oil and oleic acid content (seeExample 6) were planted for events AFS4818.1.2, AFS4818.1.3,AFS4818.1.5, AFS48182.6, AFS4818.1.9. DNA was isolated, digested withthe two restriction enzymes EcoRI and HindIII and transferred to nylonmembranes using standard protocols. Duplicate blots were produced andhybridized independently with probes corresponding to a 1.21 kbrestriction fragment of the YL DGAT1 gene (generated by digestion ofKS349 with NcoI/EcoRI) and the intact YL DGAT2 genes (generated by NotIdigestion of KS 362). Based on the sequence of KS 349 (SEQ ID NO:48) andKS 362 (SEQ ID NO: 52) insertion of an intact copy of YL DGAT1 and YLDGAT2 gene in the soybean genome would be indicated by a stronghybridization signal of restriction fragments with a size of 1.908 and3.335 kb, respectively. In keeping with this, all events showed stronglyhybridizing bands of 1.908 kb when a YL DGAT1 probe was used (FIG. 8 A).No hybridization signal was observed when DNA from unmodified soybeanswas used (lanes 11 and 12, FIGS. 8 A and B). This demonstrates that allevents tested have insertions of at least one copy of the intact YLDGAT1 expression cassette present on KS 349. However when DNA of YLDGAT2 was used in hybridization experiments, event 4818.1.9 did onlyshow a very weakly hybridizing band of high MW whereas all other eventstested showed strongly hybridizing bands of 3.335 kb (lane 9, FIG. 8B).Next genomic DNA of all five events was digested with BstXI, transferredto nylon membranes and probed with intact YL DGAT2 DNA generated asdescribed above (FIG. 9). Insertion of an intact copy of the YL DGAT2expression cassette would be indicated by strongly hybridizing bands of0.584 kb (internal fragment) and additional fragments of ≥0.2 and ≥0.77kb. All events except 4818.1.9 show the hybridization pattern indicativeof complete insertion of YL DGAT2. It was concluded that 4818.1.9 onlycontains a functional expression unit for YL DGAT1.

T1 plants of events 4818.1.9 and 4818.1.3 that were derived from seedwith increased oil and oleic content of event 4818.1.9 and 4818.1.3 weregrown to maturity and seed were harvested. Fatty acid composition of T2seed was determined by TMSH-derivatization and GC analysis of seed chipsderived from T2 seed. Table compares fatty acid composition oftransgenic and null segregant seed from a T2 plant of 4818.1.9 and4818.1.3 (Table 15B). It demonstrates that expression of YL DGAT1 aswell as co-expression of YL DGAT1 and YL DGAT2 alters the FA profile ofsoybean seed. The most pronounced alteration is an increase in oleicacid and a decrease in linolenic acid. Expression of YL DGAT genes alsoleads to a decrease in palmitic and linoleic acid and an increase instearic acid.

TABLE 15B Fatty Acid Composition of T2 Soybean Seed Generated byco-transformation with KS349 and KS362 palmitic stearic oleic linoleiclinolenic Event n acid acid acid acid acid AFS4818.1.9 58 10.8 4.5 27.949.6 7.2 average AFS4818.1.9 42 12.2 3.4 14.3 57.0 13.0 average (Nullsegregant) AFS4818.1.3 34 10 4.2 31.6 48.7 5.5 average AFS4818.1.3 1411.4 3.0 16.4 58.7 10.5 average (Null segregant)

Example 12 Analysis of Transgenic Events: Growth Chamber

The present example describes measurements of oil content of soybeanderived form T2 plants that were homozygous or heterozygous fortransgenes comprising YL DGAT1 or YL DGAT2 or both YL DGAT genes. T2plants were grown in a controlled environment (growth chamber).

Oil analysis of T2 soybean seed derived from plants grown in a plantgrowth chamber was performed by NMR. Seed were harvested form individualplants. Seed selections from heterozygous plants derived formtransformations with the DGAT2 gene from Yarrowia showed segregation ofthe DS red marker. Oil content of DS red positive seed (with DGAT2transgene) and null segregant seed from the same plant is shown in Table16. In said table oil content of seed containing the DGAT2 transgene iscompared to that of non-transgenic null segregant seed from the sameplant.

TABLE 16 Oil content of transgenic seed and null segregant seed derivedfrom transgenic soybean T2 plants that segregate for transgene with theyarrowia DGAT2 gene average aver- oil (%) age w oil (%) Δ % Event PLANTtransg. n null n points Δ % 4822.1.13 A 22.2 37 19.6 11 2.3 11.5 B 21.427 19.5 13 average 21.8 19.6 4822.4.5 A 22.3 35 18.4 13 3.6 19.6 B 22.538 18.5 10 C 21.7 31 17.7 17 D 22.7 31 18.2 17 E 21.7 23 18.9 17 average22.2 18.6 4822.1.9 A 22.9 33 20.4 7 2.5 12.3 4822.2.10 A 24.3 62 20.1 243.3 16.3 B 23 30 19.5 10 C 22.5 23 20.5 17 average 23.3 20.0

T2 seed selections of events generated by co-transformation of KS349 andKS362 were screened by GC analysis of seed chips as described above(Example 6). Seed were harvested from individual plants. Seed selectionsfrom heterozygous plants derived from transformations with Yarrowia DGATgenes segregated for elevated oleic acid content (>22% of total FA).Example 11 describes that event 4818.1.9 only contains an intactexpression cassette for the DGAT1 gene from Yarrowia. Oil content ofseed with elevated oleic acid content (with DGAT1 transgene) and nullsegregant seed from the same plant is shown in Table 17. In this table,oil content of seed containing the DGAT1 transgene was compared to thatof non-transgenic null segregant plants from the same plant.

TABLE 17 Oil content of transcenic seed and null secrecant seed derivedfrom transcenic soybean T1 plants that segregate for a transgene withthe yarrowia DGAT1 gene average oil (%) average w oil (%) Δ % EventPLANT transg. n null n points Δ % 4818.1.9 A 21.8 58 18.9 42 4.4 25.1 B21.3 16 17.0 6 C 23.1 34 17.0 14 average 22.1 17.6

Example 11 describes that other events generated by co-transformation ofKS349 and KS362 contain an intact expression cassette for both DGATgenes of Yarrowia. Oil content of seed with elevated oleic acid content(with both DGAT transgenes) and null segregant seed from the same plantis shown in Table 18. In this table, oil content of seed containing bothDGAT trangenes was compared to that of non-transgenic null segregantplants from the same plant.

TABLE 18 Oil content of transgenic seed and null segregant seed derivedfrom transgenic soybean T1 plants that segregate for transgenes withDGAT1 and DGAT2 genes from yarrowia average oil (%) average w oil (%) Δ% Event PLANT transg. n null n points Δ % 4818.1.2 A 23.9 33 20 15 3.417.3 B 22.9 35 20.3 13 C 23 31 19.2 17 average 23.3 19.8 4818.1.3 A 24.532 21.6 16 3.4 16.3 B 23.6 34 19.5 14 C 25.3 28 22 20 average 24.5 21.04818.2.6 A 22.3 31 19 17 3.2 15.8 B 24.1 21 20.6 27 C 24.1 23 20.7 25 D23.2 30 20.6 18 average 23.4 20.2

T2 seed selection homozygous for the KS362 derived Yarrowia DGAT2expression cassette no longer segregated for the DS red marker. Oilcontent of 48 seed (or all available seed if less than 48 seed wereavailable) was measured by NMR. For each event DS-red negative T1 seedwere planted and T2 seed of null segregants were harvested from plantsgrown in the same growth chamber used for cultivation of T1 plantshomozygous for the DGAT transgene. Oil content of seed derived form nullsegregant selections and lines homozygous for the DGAT transgene inshown in Table 19.

TABLE 19 Oil content of null segregant seed and transgenic seed derivedfrom transgenic soybean T1 plants that are homozygous for transgeneswith yarrowia DGAT2 gene average Δ % EVENT PLANT n oil (%) points Δ %4822.1.13 A 48 22.7 2.3 11.6 B 48 21.9 C 48 22.7 average 22.4 NULL-A 4819.7 NULL-B 48 20.5 average 20.1 4822.1.9 A 48 23.0 3.6 18.5 B 48 24.0 C48 22.7 average 23.2 NULL-A 48 19.6 4822.2.10 A 48 22.5 2.3 10.9 B 4824.4 average 23.5 NULL-A 48 20.9 NULL-B 48 21.4 average 21.2

T2 seed selection homozygous for KS349-derived Yarrowia DGAT1 andKS362-derived DGAT2 expression cassettes no longer segregated withrespect to the elevated oleic acid phenotype (22% oleic) associated withexpression of yarrowia DGAT genes. Oil content of 48 seed (or allavailable seed if less than 48 seed were available) was measured by NMR.For each event T1 null segregant seed that showed no elevation oleicacid of were planted and T2 seed of these null segregants were harvestedfrom plants grown in the same growth chamber used for cultivation of T1plants homozygous for the DGAT transgenes. Oil content of seed derivedform null segregant selections and lines homozygous for the DGATtransgene in shown in Table 20

TABLE 20 Oil content of null segregant seed and transgenic seed derivedfrom transgenic soybean T1 plants that are homozygous for transgeneswith DGAT1 and DGAT2 genes from yarrowia average Δ % EVENT PLANT n oil(%) points Δ % 4818.1.2 A 35 23.9 3.8 18.5 B 48 24.1 average 24.0 NULL-A48 20.1 NULL-B 48 20.4 average 20.3 4818.1.3 A 48 24.4 4.5 22.8 B 4824.4 C 48 24.3 average 24.4 NULL-A 48 20.2 NULL-B 48 19.5 average 19.94818.1.5 A 48 24.4 4.8 24.2 B 13 24.4 C 48 25.1 average 24.6 NULL-A 4819.4 NULL-B 48 20.2 average 19.8 4818.2.6 A 46 24.2 3.1 14.4 NULL-A 1121.4 NULL-B 48 20.9 average 21.2

In summary, growth chamber results show excellent heritability of theincreased oil trait associated with overexpression of either a singleyarrowia DGAT genes or co-expression of both yarrowia DGAT genes insoybean seed. Oil increase (compared to null segregant seed) associatedwith expression of a single yarrowia DGAT genes is at least 10.9% and ashigh as 25.1%. Oil increase (compared to null segregant seed) associatedwith expression of both yarrowia DGAT genes is at least 14.4% and ashigh as 24.2%.

Example 13A Analysis of Transgenic Events: Field

The present example describes measurements of oil content of soybeanderived from T2 plants that were homozygous or heterozygous fortransgenes comprising YL DGAT1 or YL DGAT2 or both YL DGAT genes. T2plants were grown in a non-controlled environment (field).

DS red positive T1 seed of transgenic events generated with YL DGAT2(contained in KS 362) and corresponding DS red negative null segregantseed were grown in a field in Iowa in the summer of 2007. T1 seed withelevated oleic acid content that had been generated by co-transformationwith YL DGAT1 and YL DGAT2 and corresponding null segregant seed withnormal levels of oleic acid were grown in a similar fashion. T2 seedwere harvested from individual plants and subjected to NMR analysis tomeasure oil content. Table 21 shows oil content of 48 uniformly DS redpositive seed derived from events that were homozygous for the KS362transgene and that of DS-red negative seed from null segregant seed ofthe same event grown in the same environment.

TABLE 21 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are homozygous fortransgenes with the yarrowia DGAT2 gene average Δ % EVENT PLANT n oil(%) points Δ % 4822.1.2 A 48 22.4 2.4 13 B 48 20.5 C 48 21.5 E 48 21.7 F48 20.7 G 40 19.5 H 16 23 average 21.3 Null A 48 18.9 Null B 48 19 NullC 48 18.7 Null D 16 17.9 Null E 24 20.2 average 18.9 4822.2.11 A 48 22.12.6 14 B 47 21 C 48 23.8 D 48 21.1 E 48 23.1 F 48 22 G 48 20.4 H 48 20.8I 48 20.3 average 21.6 Null A 48 19.2 Null B 48 19.5 Null C 48 18.3average 19.0 4822.2.10 A 40 21.8 2.6 13.6 B 48 21.8 average 21.8 Null A48 18.6 Null B 48 19.2 Null C 48 19.2 Null E 16 19.7 Null F 48 19.7 NullG 48 19 average 19.2

Table 22 shows oil content of 48 seed from segregants that werehomozygous for YL DGAT1 and YL DGAT2 transgenes. All seed harvested fromthese homozygous T2 seed selections showed the elevated oleic acidcontent associated with YL DGAT expression. In Table 22 oil content ofthese lines is compared to that of null segregant seed of the sameevents with unaltered levels of oleic acid, derived from plants grown inthe same environment.

TABLE 22 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are homozygous fortransgenes with the yarrowia DGAT1 and DGAT2 genes. average Δ % EVENTPLANT n oil (%) points Δ % 4818.1.2 A 37 22.2 3.8 20 B 48 22.5 C 48 22.7D 48 22.6 E 48 24.3 F 40 21.5 average 22.6 Null A 48 17.9 Null B 48 18.9Null C 48 19.4 Null D 48 19.5 Null E 48 19.7 Null F 48 18.4 Null G 4817.9 average 18.8 4818.1.3 A 48 23.5 3.5 19 B 48 22.4 C 48 22.8 D 1921.3 E 48 20.5 F 48 22.3 G 48 22.8 H 48 22.5 average 22.3 Null A 48 18.4Null B 48 19.1 Null C 48 17.9 Null D 48 19 Null E 48 19.3 Null F 48 18.3Null G 48 18.3 average 18.6

Example 11 describes that event 4818.1.9 only contains an intactexpression cassette for the DGAT1 gene from Yarrowia. Three T1 plants ofthis event derived from T1 seed with elevated oleic acid content weregrown in the Iowa field along side null segregant plants derived from T1seed with unaltered oleic acid content. T2 seed from all threetransgenic segregants still showed segregation of the elevated oleicacid phenotype indicating that the parental lines were stillheterozygous for the DGAT1 transgene. Using GC analysis alltransgene-positive seed were identified from these lines and subjectedto oil analysis by NMR. In Table 23 oil content of these seed iscompared to that of null segregant seed derived from T1 plants gown inthe same environment.

TABLE 23 Oil content null segregant seed and transgenic seed derivedfrom field-grown transgenic soybean T1 plants that are heterozygous fortransgenes with the yarrowia DGAT1 gene. average Δ % EVENT PLANT n oil(%) points Δ % 4818.1.9 A 14 20.7 2.0 11 B 14 19.1 C 20 20 average 20.0Null A 40 18.1 Null B 40 17.8 average 18.0

In summary, field environment results show excellent heritability of theincreased oil trait associated with overexpression of either a singleyarrowia DGAT genes, or co-expression of both yarrowia DGAT genes insoybean seed. Oil increase (compared to null segregant seed) associatedwith expression of a single yarrowia DGAT genes is at least 11% and ashigh as 14%. Oil increase (compared to null segregant seed) associatedwith expression of both yarrowia DGAT genes is at least 19% and as highas 20%.

Example 13B Compositional Analysis of Soybean Seed

The present example describes measurements of seed compositionalparameters such as protein content and content of soluble carbohydratesof soybean seed derived from transgenic events that express single YLDGAT genes (YL DGAT2) of both YL DGAT genes.

Changes in the composition of soybean seed associated with expression ofYL DGAT genes were measured. To this end the concentrations of protein,soluble carbohydrates and starch were measured as follows.

Non-Structural Carbohydrate and Protein Analysis.

Dry soybean seed were ground to a fine powder in a GenoGrinder andsubsamples were weighed (to an accuracy of 0.1 mg) into 13×100 mm glasstubes; the tubes had Teflon® lined screw-cap closures. Three replicateswere prepared for each sample tested. Tissue dry weights were calculatedby weighing sub-samples before and after drying in a forced air oven for18 h at 105 C.

Lipid extraction was performed by adding 2 ml aliquots of heptane toeach tube. The tubes were vortex mixed and placed into an ultrasonicbath (VWR Scientific Model 750D) filled with water heated to 60 C. Thesamples were sonicated at full-power (˜360 W) for 15 min and were thencentrifuged (5 min×1700 g). The supernatants were transferred to clean13×100 mm glass tubes and the pellets were extracted 2 more times withheptane (2 ml, second extraction, 1 ml third extraction) with thesupernatants from each extraction being pooled. After lipid extraction 1ml acetone was added to the pellets and after vortex mixing, to fullydisperse the material, they were taken to dryness in a Speedvac.

Non-Structural Carbohydrate Extraction and Analysis.

Two ml of 80% ethanol was added to the dried pellets from above. Thesamples were thoroughly vortex mixed until the plant material was fullydispersed in the solvent prior to sonication at 60 C for 15 min. Aftercentrifugation, 5 min×1700 g, the supernatants were decanted into clean13×100 mm glass tubes. Two more extractions with 80% ethanol wereperformed and the supernatants from each were pooled. The extractedpellets were suspended in acetone and dried (as above). An internalstandard β-phenyl glucopyranoside (100 ul of a 0.5000+/−0.0010 g/100 mlstock) was added to each extract prior to drying in a Speedvac. Theextracts were maintained in a desiccator until further analysis.

The acetone dried powders from above were suspended in 0.9 ml MOPS(3-N[Morpholino]propane-sulfonic acid; 50 mM, 5 mM CaCl₂), pH 7.0)buffer containing 1000 of heat stable α-amylase (from Bacilluslicheniformis; Sigma A-4551). Samples were placed in a heat block (90 C)for 75 min and were vortex mixed every 15 min. Samples were then allowedto cool to room temperature and 0.6 ml acetate buffer (285 mM, pH 4.5)containing 5 U amyloglucosidase (Roche 110 202 367 001) was added toeach. Samples were incubated for 15-18 h at 55 C in a water bath fittedwith a reciprocating shaker; standards of soluble potato starch (SigmaS-2630) were included to ensure that starch digestion went tocompletion.

Post-digestion the released carbohydrates were extracted prior toanalysis. Absolute ethanol (6 ml) was added to each tube and aftervortex mixing the samples were sonicated for 15 min at 60 C. Sampleswere centrifuged (5 min×1700 g) and the supernatants were decanted intoclean 13×100 mm glass tubes. The pellets were extracted 2 more timeswith 3 ml of 80% ethanol and the resulting supernatants were pooled.Internal standard (100 ul β-phenyl glucopyranoside, as above) was addedto each sample prior to drying in a Speedvac.

Sample Preparation and Analysis

The dried samples from the soluble and starch extractions describedabove were solubilized in anhydrous pyridine (Sigma-Aldrich P57506)containing 30 mg/ml of hydroxylamine HCl (Sigma-Aldrich 159417). Sampleswere placed on an orbital shaker (300 rpm) overnight and were thenheated for 1 hr (75 C) with vigorous vortex mixing applied every 15 min.After cooling to room temperature 1 ml hexamethyldisilazane(Sigma-Aldrich H-4875) and 100 ul trifluoroacetic acid (Sigma-AldrichT-6508) were added. The samples were vortex mixed and the precipitateswere allowed to settle prior to transferring the supernatants to GCsample vials.

Samples were analyzed on an Agilent 6890 gas chromatograph fitted with aDB-17MS capillary column (15 m×0.32 mm×0.25 um film). Inlet and detectortemperatures were both 275 C. After injection (2 ul, 20:1 split) theinitial column temperature (150 C) was increased to 180 C at a rate 3C/min and then at 25 C/min to a final temperature of 320 C. The finaltemperature was maintained for 10 min. The carrier gas was H2 at alinear velocity of 51 cm/sec. Detection was by flame ionization. Dataanalysis was performed using Agilent ChemStation software. Each sugarwas quantified relative to the internal standard and detector responseswere applied for each individual carbohydrate (calculated from standardsrun with each set of samples). Final carbohydrate concentrations wereexpressed on a tissue dry weight basis.

Protein Analysis

Protein contents were estimated by combustion analysis on a ThermoFinnigan Flash 1112EA combustion analyzer. Samples, 4-8 mg, weighed toan accuracy of 0.001 mg on a Mettler-Toledo MX5 micro balance were usedfor analysis. Protein contents were calculated by multiplying % N,determined by the analyzer, by 6.25. Final protein contents wereexpressed on a % tissue dry weight basis.

TABLE 24 Compositional analysis of soybean seed derived from two T1plants that were either a null segregant or homozygous for an YL DGAT1YL DGAT2 transgene. The plants were grown in the same growth chamberenvironment. If not indicated otherwise values are reported as g/kg DW.Myo- Total Protein Event Pinitol Sorbitol Fructose Glucose InositolSucrose Raffinose Stachyose g/kg Starch (% DW) 4818.1.5 Mean 1.57 0.264.60 1.61 0.27 47.0 8.01 35.88 99.2 0.45 38.6 NULL SD 0.03 0.02 0.180.27 0.01 1.6 0.15 0.72 2.0 0.03 0.3 4818.1.5 Mean 1.31 0.24 2.67 0.790.31 21.2 5.08 32.60 64.2 0.03 43.4 TG SD 0.09 0.00 0.07 0.02 0.01 0.40.11 1.21 1.7 0.02 0.2

TABLE 25 Compositional analysis of soybean seed derived from a T1 plantthat was heterozygous for an YL DGAT2 transgene. The plant was grown ina growth chamber. YL DGAT transgenic seed and null segregant seed wereselected based on DS red expression as a visible marker. Eight DS redpositive and DS red- negative seed were combined and analyzed asdescribed above. If not indicated otherwise values are reported as g/kgDW. Protein Myo- Total (% Event Pinitol Sorbitol Fructose GlucoseInositol Sucrose Raffinose Stachyose g/kg Starch DW) 4822.2.10 Mean 1.660.17 5.50 2.57 0.28 47.2 6.64 45.0 108.9 0.31 40.2 NULL SD 0.12 0.041.76 0.87 0.09 0.2 0.68 1.6 3.1 0.05 0.7 4822.2.10 Mean 2.02 0.19 5.021.72 0.33 32.7 6.91 41.4 90.3 0.31 40.8 TG SD 0.06 0.01 0.33 0.18 0.021.0 0.18 0.6 1.5 0.11 0.9

TABLE 26 Compositional analysis of soybean seed derived from T1 plantsthat were heterozygous for a YL DGAT2 or a YL DGAT1 YL DGAT2 transgene.The plants were grown in the field. YL DGAT transgenic seed and nullsegregant seed were selected based on DS red expression (4822.2.10) as avisible marker or elevated oleic acid content determined by GC analysis(4818.1.2). Eight transgene-positive and transgene-negative seed werecombined and analyzed as described above. If not indicated otherwisevalues are reported as g/kg DW. Myo- Total Protein Event PinitolSorbitol Fructose Glucose Inositol Sucrose Raffinose Stachyose g/kgStarch (% DW) 4822.2.10 Mean 2.44 0.26 1.16 1.12 0.44 57.7 8.5 39.0110.6 3.20 41.5 NULL SD 0.05 0.01 0.05 0.02 0.00 0.7 0.4 0.5 1.3 0.550.5 4822.2.10 Mean 1.89 0.35 0.82 0.67 0.32 29.7 5.5 31.7 70.9 1.00 46.1TG SD 0.06 0.09 0.14 0.06 0.01 1.5 0.1 0.5 2.2 0.23 0.7 4818.1.2 Mean1.79 0.51 1.17 1.12 0.37 49.8 6.1 42.0 102.8 0.51 44.5 NULL SD 0.02 0.010.13 0.13 0.01 2.6 0.4 2.4 5.1 0.07 2.2 4818.1.2 Mean 1.87 0.32 0.710.52 0.40 35.7 6.0 37.7 83.2 0.35 46.4 TG SD 0.05 0.02 0.05 0.01 0.020.8 0.2 1.5 2.5 0.08 0.2

Tables 24-26 illustrate that expression one or two YL DGAT genes indifferent environments (growth chamber, field) is associated with aconsistent shift in seed composition that is characterized by areduction in soluble carbohydrates, namely a reduction in sucrose and toa smaller extent a reduction in stachyose. Most importantly there is noreduction in protein content observed when oil accumulation is increasedthrough expression of YL DGAT genes.

Example 14 Measurements of DGAT Activity in Developing Seed and SomaticEmbryos

The present example describes construction of soybean expression vectorscomprising Yarrowia DGAT2 alone or Yarrowia DGAT2 and DGAT1, expressionof these gene(s) in soybean seed or somatic embryos and DGAT enzymeactivity in these tissues.

Construction of pKR1234 comprising YL DGAT2

The NotI fragment of KS362 (SEQ ID NO:52), containing the YL DGAT2, wascloned into the NotI fragment of pKR72 (SEQ ID NO:26; Example 4) toproduce pKR1234 (SEQ ID NO:68).

Construction of pKR1236 Comprising YL DGAT1 and DGAT2

The glycinin Gy1 promoter was PCR amplified from pZBL119 (which isdescribed in PCT Publication No. WO 2004/071467 and the contents ofwhich are hereby incorporated by reference) using primers oSGly-2 (SEQID NO:69) and oSGly-3 (SEQ ID NO:70). The resulting PCR fragment wassubcloned into the intermediate cloning vector pCR-Script AMP SK(+)(Stratagene), according to the manufacturer's protocol, to produceplasmid pPSgly32 (SEQ ID NO:71).

The PstI/NotI fragment of plasmid pSGly32 (SEQ ID NO:71), containing theGy1 promoter, was cloned into the PstI/NotI fragment from plasmid pKR142(which is described in PCT Publication No. WO 2004/071467), containingthe leguminA2 3′ transcription termination region, an ampicillinresistance gene, and bacterial ori, to produce pKR264 (SEQ ID NO:72).Thus, vector pKR264 contains a NotI site flanked by the promoter for theglycinin Gy1 gene and the leguminA2 3′ transcription termination region(Gy1/NotI/legA2 cassette).

The NcoI/XbaI fragment of KS349 (SEQ ID NO:48), containing YarrowiaDGAT1, was cloned into the NcoI/XbaI sites of pKR908, (US Pat. Pub.20080095915), which contains NcoI/XbaI sites flanked by NotI sites, toproduce pKR1212 (SEQ ID NO:73).

The NotI fragment of pKR1212 (SEQ ID NO:73), containing the YarrowiaDGAT1 gene, was cloned into the NotI site of pKR264 (SEQ ID NO:72) toproduce pKR1235 (SEQ ID NO:74).

The BsiWI fragment of pKR1235 (74), containing the Yarrowia DGAT1, genewas cloned into the BsiWI site of pKR1234 (SEQ ID NO:68) to producepKR1236 (SEQ ID NO:75).

DGAT Assays on Microsomal Extracts from Developing T2 Seed

Soybean embryogenic suspension cultures (cv. Jack) were transformed withKS362 as described herein (Example 6), comprising Yarrowia DGAT2, asdescribed herein and T1 seed from soy plants AFS4822.1.13.1 (seed called7GR11-58) and AFS4822.2.10.1 (seed called 7GR11-66) were planted andplants grown as described in Example 12. In both of these events seedoil concentration of transgene positive seed was found to be elevatedwhen compared to null segregant seed of the same event.

Similarly, soybean embryogenic suspension cultures (cv. Jack) wereco-transformed with KS362 (comprising YL DGAT2) and KS349 (comprising YLDGAT1) as described herein (Example 6). Transgene-positive T1 seed fromevent AFS4818.1.2.1 are represented by seed 7GR11-2. In this event, seedoil concentration of transgene positive seed was found to be elevatedwhen compared to null segregant seed of the same event (Example 6).Transgene-negative, null segregant T1 seed derived from AFS4818.1.2.1and AFS4818.1.3.1 are represented by 7GR11-7 and 7GR11-15. These seedwere planted and plants grown as described in Example 12.

Approximately 1 g of T2 seed were collected from selected plants 30 daysafter flowering (DAF) and were snap frozen in liquid nitrogen and storedat −80 C until ready to process. After grinding 1 g of frozen seedtissue in liquid nitrogen in a mortar and pestle, 3 mL of planthomogenization buffer (300 mM sucrose; 1 mM EDTA; 10 mM Tris.HCl, pH8.0; 1 mM DTT; 0.1% polyvinylpolypyrrolidine) was added and tissue wasfurther homogenized using a polytron homogenizer for 1 minute. Debriswas collected by vacuum filtration through 3 layers of cheese clothfollowed by filtration through 1 layer of mira cloth. The resultingfiltrate was centrifuged for 15 min. twice at 1,500×g and the resultingsupernatant was then centrifuged at 100,000×g for 60 min. The resultingpellet was responded in approximately 0.5 to 1 mL of microsome buffer(100 mM potassium phosphate, pH 7.2) by gentle pipetting followed byfurther resuspension in a 2 mL sized Teflon-coated Dounce homogenizer.Protein concentrations were determine using Bradford reagent(Sigma-Aldrich) and microsomes were snap frozen in liquid nitrogen andstored at −80 C until assayed.

DGAT assays were carried out for 5 min at 25° C. in plant assay buffer(500 mM Tricine, pH 7.8; 28 mM sodium chloride; 0.06% CHAPS), with 20 μM1-14 C-labeled oleoyl-coenzyme A (50 mCi/mmol, Perkin Elmer), 1.5 mMdioleoylglyceride (Sigma-Aldrich) and 20 μg of microsomal protein in atotal reaction volume of 100 μl. Each reaction was initiated by additionof the microsomal membranes to the remainder of the reaction components.Assays were terminated by the addition of 1 mL of hot isopropanol (75 C)and heating at 75 for 10 min. Assays were cooled to RT, 1.5 mL of hexanewas added and samples were mixed. Phases were separated by low speedcentrifugation after addition of 1.25 ml of 500 mM sodium sulfate andthe upper phase was transferred to another glass tube. The top phase wasthen dried under nitrogen gas. The lipid from each assay wasre-dissolved in 75 uL of hexane spiked with 1 uL of soybean oil. Lipidwas applied to a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 umthickness, 20 cm×20 cm) and triglycerides were separated from otherlipids by development with 80:20:1 (v/v/v) hexane:diethylether:aceticacid. Triacylglycerol was visualized and marked by light staining iniodine vapor in a tank. The plate was removed from the iodine tank andafter the stain faded, the triacylglycerol was scraped, andradioactivity determined by liquid scintillation counting and expressedas dpm per min. Total activity was determined as the amount ofradiolabeled oleic acid incorporated into triacylglycerol per minute permg of protein using the following formula: ([dpm]/[2200000 dpm/uCi]/[50uCi/umol]/[5 min.]/[0.02 mg protein]×[1000 nmol/umol]). DGAT activitiesfor each of the samples described are shown in Table 27.

TABLE 27 DGAT activities for selected developing T2 seed T2 DevelopingT1 Seed Seed DGAT T1 Oil Activity Exp. Plasmid DGAT Event T1 plant SeedPhenotype (nmol.min-1.mg-1) 4818 KS362 DGAT2 AFS4818.1.2 AFS4818.1.2.17GR DGAT 2.8 KS349 DAGT1 11-2 4818 KS362 DGAT2 AFS4818.1.2 AFS4818.1.2.17GR null 0.4 KS349 DAGT1 11-7 4818 KS362 DGAT2 AFS4818.1.3 AFS4818.1.3.17GR null 0.4 KS349 DAGT1 11-15 4822 KS362 DGAT2 AFS4822.1.13AFS4822.1.13.1 7GR DGAT 2.4 11-58 4822 KS362 DGAT2 AFS4822.2.10AFS4822.2.10.1 7GR DGAT 1.6 11-66DGAT Assays on Microsomal Extracts from Soybean Somatic Embryos

Soybean embryogenic suspension cultures (cv. Jack) were transformed witheither pKR1234 (SEQ ID NO:68), comprising Yarrowia DGAT2 and havingexperiment number MSE2181, or with pKR1236 (SEQ ID NO:75), comprisingYarrowia DGAT2 and DGAT1 and having experiment number MSE2182. Eventswere selected and somatic embryos matured in SHaM as described inExample 5. After 2 weeks of maturation in SHaM, approximately 1 g oftissue from each event was frozen in liquid nitrogen and tissue wasground with a mortar and pestle as described for soybean developingseed. A small amount of ground tissue (approximately 100 mg) waslyophilized overnight and the remaining tissue was stored at −80 C.

Oil concentrations were determined on approximately 10 mg of lyophilizedtissue from each event using the GC method and 17:0 internal standardexactly as described in Example 5 and results for oil concentrations andoleic acid content (% of total FAME) are shown in Table 28. Microsomalprotein preparations were made, protein concentrations determined andDGAT assays were carried out on selected events determined to have arange of oil concentrations exactly as previously described for seedtissue. Results for DGAT assays are also shown in Table 28.

TABLE 28 Esterified Fatty Acid, Oleic Acid Content and DGAT activitiesof Soybean Somatic Embryos Transformed with either pKR1234 or pKR1236oleic acid FAME (% total DGAT Activity Event # (% DCW) FAME) (nmol · min− 1 · mg − 1) MSE2181-pKR1234 (YL DGAT2) 8 8.9 31.9 3.6 5 7.3 33.8 4.5 36.6 32.6 2.5 9 6.2 30.7 2.5 7 6.1 30.4 16 5.8 25.4 1.6 12 5.6 26.1 155.4 26.7 2.8 4 4.6 22.5 0.7 11 4.2 22.7 0.5 14 4.0 21.2 13 3.8 23.2 1.86 3.5 25.3 10 3.2 21.2 0.5 MSE2182-pKR1236 (YL DGAT2/YL DGAT1) 2 10.938.8 6.3 7 10.8 38.8 4 10.3 38.4 5.1 3 10.0 38.0 8 9.8 38.1 4.4 17 9.629.3 10 8.6 33.3 5.7 28 8.2 36.3 13 8.1 43.6 5 8.0 25.4 20 8.0 36.4 127.7 29.3 4.8 26 7.6 30.5 24 7.2 33.4 9 6.3 20.4 0.7 29 6.3 29.8 21 6.131.1 19 5.7 32.5 6 5.6 21.7 0.5 23 5.6 33.5 16 5.4 26.3 25 5.1 20.3 145.1 26.2 0.9 1 5.0 24.9 18 4.9 22.2 30 4.5 17.0 0.4 27 3.7 19.7 15 3.221.6 11 2.9 16.6 2.5 22 2.8 20.7

Events transformed with pKR1234 and having some of the highest oilconcentrations had increases in DGAT activity of up to 9-fold comparedwith those events having wild-type levels of oil. Events transformedwith pKR1236 and having some of the highest oil concentrations hadincreases in DGAT activity of up to 15.8-fold compared with those eventshaving wild-type levels of oil.

Soybean embryogenic suspension culture (cv. Jack) was also transformedwith KS364 (SEQ ID NO:63), comprising Yarrowia DGAT2 and DGAT1(Experiment # MSE2134) and individual events were analyzed for fattyacid profile and oil concentration as described in Example 5. Based onthis data, one event (Event 54) having high oleic acid (32.83% of totalfatty acids) and oil concentrations (12.5% DCWt) and one event (Event33) having wild-type levels of oleic acid (15.52% of total fatty acids)and oil concentrations (8.2% DCWt) were chosen for DGAT assays.Transformed embryogenic suspension culture from each event was bulked upin SB 196 media and embryos matured in SHaM as described in Example 5.

After 2 weeks of maturation in SHaM, approximately 1 g of tissue fromeach event was frozen in liquid nitrogen, microsomal proteinpreparations were made and DGAT assays were carried out on each eventexactly as previously described and results are shown in Table 29. Totallipid was also extracted and oleic acid and oil concentrations weredetermined as described below and results are reported in Table 29.

TABLE 29 Esterified Fatty Acid, Oleic Acid Content and DGAT activitiesof Soybean Somatic Embryos Transformed with KS364 KS364 (DGAT2/DGAT1)oleic acid FAME (% total DGAT Activity Event # (% DCW) FAME) (nmol · min− 1 · mg − 1) 33 8.2 15.5 0.3 54 12.7 32.1 3.2

The event having the high oil concentration (Event 54) had increases inDGAT activity of 10.7-fold compared with the event having wild-typelevels of oil (Event 33).

Example 15 Analysis of Lipid Fractions of Transgenic Seed and SomaticEmbryos Expressing DGAT Genes

Soy somatic embryos transformed with KS364 from an event with wild-typeconcentrations of oil and oleic acid (Event 33) and from an event withhigh concentrations of oil and oleic acid (Event 54) were lyophilizedfor 48 hr and tissue was ground using a genogrinder exactly as describedin Example 5.

Total Lipid Extraction

Total lipid was extracted from each event by the method of Bligh, E. G.& Dyer, W. J. (Can. J. Biochem. Physiol. 37:911-917 (1959)) with somemodifications. Briefly, approximately 100 mg of ground tissue from eachevent was added to a 16 mm×125 mm sized test-tube with a teflon-linedscrew cap lid. A mixture of methanol:chloroform/2:1 (6 mL) was added andthe sample was extracted with gentle mixing for 1 hr after which 2 mL ofchloroform was added followed by continued mixing for 30 min.Afterwards, 3.6 mL of water was added, the tube was vortexed vigorouslyand phases were separated by centrifugation in a clinical centrifuge.The lower organic layer was gently removed to a second glass test tubeand the upper aqueous layers were re-extracted with 2 mL of chloroform.Centrifugation was repeated and the lower organic phase was combinedwith the first organic phase. Samples were dried under a stream ofnitrogen at 50 C, total lipid was estimated by weighing and lipid wasdissolved in chloroform:methanol/6:1 to a concentration of approximately10 mg/mL. FAME analysis was carried out on approximately 50 ug of eachsample using the sulfuric acid/methanol procedure described herein(Example 4) and results are shown in Table 30.

Separation of Neutral and Polar Lipids

Sep-pak amino-propyl solid phase extraction columns (Waters; 6 cccolumns, WAT054560) were equilibrated with 5 mL of methanol followed by5 mL of methanol:chloroform/1:1 followed by 5 mL of chloroform.Approximately 5 mg of total lipid in chloroform:methanol/6:1 was addedto each column, followed by 5×1 mL aliquots of chloroform to eluteneutral lipids and all fractions were collected, combined and driedunder a stream of nitrogen at 50 C. Polar lipids were then eluted fromeach column using 5×1 mL aliquots of methanol:chloroform/1:1 followed by5×1 mL aliquots of methanol and all fractions were combined and driedunder nitrogen. Neutral lipids were dissolved in approximately 1 mL ofCHCl3:MeOH/6:1 and polar lipids were dissolved in approximately 200 uLof CHCl3:MeOH/6:1. FAME analysis was carried out on approximately 50 ugof neutral lipid using the sulfuric acid/methanol procedure describedherein (Example 4) and results are shown in Table 30. Separation of TAG,PC and PE by TLC

Approximately 100 uL of neutral lipid extract was loaded 2 cm from thebottom of a Partisil K6 Silica Gel 60 A TLC plate (Whatman, 250 umthickness, 20 cm×20 cm). Similarly, approximately 200 uL of the polarlipid fraction was loaded onto the same TLC plate. Standard solutions(10 mg/mL in chloroform:methanol/6:1) of TAG, PC and PE were alsospotted onto the plates. TLC plates were developed inCHCl3:MeOH:AcOH/65:35:8 until solvent front was approximately half wayup the plate. TLC plates were then air dried for 10 min and developedfully in 70:30:1 (v/v/v) hexane:diethylether:acetic acid. Standards werevisualized by light staining with iodine vapour and corresponding bandsfor TAG, PC and PE were cut out of the TLC plate. Silica gel containingeach lipid species was derivatized directly with sulfuric acid/methanolas described herein (Example 4) and results are shown in Table 30.

Fatty Acid Positional Analysis of TAG

Fatty acid profiles of the sn2 position of TAG were determined usingporcine pancreatic lipase to remove acyl groups from the sn1 and sn3position of TAG only, followed by transesterification of the resultingmonoacylglyceride (MAG) produced. Approximately 5 mg of neutral lipidextract was suspended in 2 mL of 1M Tris.HCl, pH 8.0 along with 0.2 mLof 2.2% calcium chloride and 0.5 mL of 0.05% Bile salts in a glass screwcap test tube. The lipid was incubated at 37 C for 5 min, 5 mg ofporcine pancreatic lipase was added directly and the suspension wasincubated with shaking at 37 C for 20 min. After incubation, thereaction was reaction was terminated with the addition of 1 mL ofethanol followed by 1 mL of 6 M HCl. After mixing, 2.5 mL of diethylether was added, phases were separated by centrifugation and the toporganic layer was removed carefully. The diethyl ether extraction wasrepeated and the top diethyl ether phase was combined with the first.After drying over anhydrous sodium sulfate, the diethyl ether wasevaporated under a stream of nitrogen at 50 C and the resulting lipidwas dissolved in 200 uL of chloroform:methanol/6:1. The lipid was loadedonto a Partisil K6 TLC plate along with triacylglyceride (TAG),diacylglyceride (DAG), monoacylglyceride (MAG) and free fatty acid (FFA)standards and the TLC plate was developed as described herein.Afterwards, standards were visualized with light iodine staining and theMAG band was cut and derivatized with methanol/sulfuric acid aspreviously described herein. Results for the fatty acid profile of FAMEfrom the MAG band, representing the fatty acid profile of the sn2position of TAG (i.e. the acyl group on C2 of glycerol), along with thecalculated sn1 and sn3 positions, is shown in Table 30. In Table 30, the% of total fatty acid for each fatty acid (i.e. 16:0, 18:0, 18:1, 18:2,18:3) at the sn1 and sn3 positions of TAG is calculated with thefollowing formula: =([TAGx]-[sn2×]/3)*3/2; where the x indicates thefatty acid of interest.

TABLE 30 Fatty acid composition of various lipid species and positionaldistribution in TAG % Sample Event Oil 16:0 18:0 18:1 18:2 18:3 Total 338.2 15.6 3.9 15.5 50.8 14.2 Extract 54 12.7 10.5 4.7 32.1 43.9 8.8Neutral 33 8.2 14.4 3.7 17.0 52.5 12.4 lipids 54 12.7 9.8 5.3 36.0 42.36.6 TAG 33 8.2 18.2 4.8 20.8 47.4 8.7 54 12.7 11.8 5.8 40.7 37.0 4.6 PC33 8.2 34.1 10.6 17.2 33.8 4.3 54 12.7 21.5 10.0 32.1 33.7 2.7 PE 33 8.241.1 6.8 13.4 25.9 12.8 54 12.7 45.8 9.8 21.9 15.3 7.1 TAG-sn1 33 8.21.0 0.3 14.9 72.7 11.0 54 12.7 1.0 0.5 24.0 67.1 7.4 TAG-sn1, 3 33 8.226.8 7.0 23.8 34.9 7.5 (Calculated) 54 12.7 17.3 8.5 49.1 22.0 3.2Changes in fatty acid profiles associated with YL DGAT expressionobserved in TAG are also observed in polar lipids.

Example 16 Yarrowia DGAT Variants with Altered Amino Acid Sequence

The present example describes the creation of mutant forms of YarrowiaDGAT2, cloning them into a yeast expression vector and assayingmicrosomal protein fractions for DGAT activity.

Constructing Saccharomyces Expression Vectors Containing Mutant YarrowiaDGAT2s

Yarrowia DGAT2 was amplified from pKR1234 (SEQ ID NO:68; Example 14)with oligonucleotide primers oYDG2-1 (SEQ ID NO:76) and oYDG2-2 (SEQ IDNO:77), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No. F553S,Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pKR1254 (SEQ IDNO:78).

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid Y326 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesYID2_Y326F-5 (SEQ ID NO:79) and YID2_Y326F-3 (SEQ ID NO:80), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that Y326 was changed to F326, was chosen for furtherstudy. This clone was designated pKR1254_Y326F (SEQ ID NO:81). Thenucleotide sequence for altered coding sequence (YIDGAT2_Y326F) is setforth in SEQ ID NO:82 and the corresponding amino acid sequence is setforth in SEQ ID NO:83.

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid Y326 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesYID2_Y326L-5 (SEQ ID NO:84) and YID2_Y326L-3 (SEQ ID NO:85), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that Y326 was changed to L326, was chosen for furtherstudy. This clone was designated pKR1254_Y326L (SEQ ID NO:86). Thenucleotide sequence for altered coding sequence (YIDGAT2_Y326L) is setforth in SEQ ID NO:87 and the corresponding amino acid sequence is setforth in SEQ ID NO:88.

A single codon in the Yarrowia DGAT2 sequence, which codes for aminoacid R327 in the corresponding amino acid sequence SEQ ID NO:10, waschanged using the Quickchange® Site Directed Mutagenesis kit (Cat. No.200518, Stratagene, La Jolla, Calif.), with oligonucleotidesYID2_R327K-5 (SEQ ID NO:89) and YID2_R327K-3 (SEQ ID NO:90), followingthe manufacturer's protocol. After extensive sequencing, a clone codingfor an amino acid sequence which is identical to the Yarrowia DGAT2 (SEQID NO:10), except that R327 was changed to K₃₂₇, was chosen for furtherstudy. This clone was designated pKR1254_R327K (SEQ ID NO:91). Thenucleotide sequence for altered coding sequence (YIDGAT2_Y326L) is setforth in SEQ ID NO:92 and the corresponding amino acid sequence is setforth in SEQ ID NO:93.

The NotI fragments of pKR1254, pKR1254_Y326F, pKR1254_Y326L orpKR1254_R327K, each containing a wild-type or mutant version of YIDGAT2,were cloned into the NotI site of pY75 (SEQ ID NO:3; Example 1) toproduce pY191 (SEQ ID NO:94), pY192 (SEQ ID NO:95), pY193 (SEQ ID NO:96)or pY194 (SEQ ID NO:97), respectively.

Assaying DGAT Activity of Mutant Yarrowia DGAT2s

A mutant strain of Saccharomyces cerevisiae where the endogenous DGAT2gene (DGA1) was knocked out and has the following genotype (BY4741, MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was obtained from Open Biosystems(http://www.openbiosystems.com/). It was transformed with pY191, pY192,pY193 or pY194 and transformants were isolated as described herein.Three individual transformants per transformation were inoculated into 2mL cultures of DOBA media supplemented with CSM-leu 30° C. for 16 h.Cells (1 mL) were transferred to 50 mL of DOBA medium described aboveand grown at 30° C. for an additional 16 h. Cells were pelleted bycentrifugation, frozen in liquid nitrogen and stored at −80° C. untilrequired for use.

Pellets were re-suspended in 2 mL of yeast homogenization buffer (20 mMTris.HCl, pH 8.0; 10 mM MgCl₂; 1 mM EDTA; 5% glycerol; 1 mM DTT; 0.3 M(NH₄)₂SO₄) and the suspension was added to a 2 mL screw cap tubecontaining approximately 1 mL of 0.5 mm glass beads. The after removalof air pockets by vortexing, the resuspension was filled to the top ofthe tube, the tube capped and the cells broken with three, 1 min. pulsesin a mini bead beater at 5000 rpm with storage on ice for 5 min. Theyeast homogenate was centrifuged at 1,500×g for 15 min. at 4 C and theresulting supernatant was then centrifuged at 100,000×g for 60 min. Theresulting pellet was responded in approximately 0.2 to 0.5 mL ofmicrosome buffer (100 mM potassium phosphate, pH 7.2) by gentlepipetting followed by further resuspension in a 2 mL sized Teflon-coatedDounce homogenizer. Protein concentrations were determine using Bradfordreagent (Sigma-Aldrich) and microsomes were snap frozen in liquidnitrogen and stored at −80 C until assayed.

DGAT assays were carried out for 1 min at 25° C. in yeast assay buffer(50 mM potassium phosphate (pH 7.2)), with 20 μM 1-14 C-labeledoleoyl-coenzyme A (50 mCi/mmol, Perkin Elmer), and 20 μg of microsomalprotein in a total reaction volume of 100 Each reaction was initiated byaddition of the microsomal membranes to the remainder of the reactioncomponents. Assays were terminated and radioactivity into TAG determinedexactly as described for the plant DGAT assays except the formula waschanged to reflect a 1 min. assay time (i.e. [dpm]/[2200000 dpm/uCi]/[50uCi/umol]/[5 min.]/[0.02 mg protein]×[1000 nmol/umol]). DGAT activitiesfor each of the samples as well as the averages described are shown inTable 31.

TABLE 31 DGAT activities for DGA1 Transformed with pY191, pY192, pY193or pY194. Avg. DGAT Activity Std. Plasmid Mutant (nmol · min⁻¹ · mg⁻¹)(nmol · min⁻¹ · mg⁻¹) Dev. pY191 wt 7.6 10.4 2.7 10.6 13.0 pY192 Y326F6.5 8.3 1.7 8.4 10.0 pY193 Y326L 6.2 8.1 1.9 10.0 8.2 pY194 R327K 5.96.2 0.4 6.0 6.7From Table 31, it appears that the Y326F and Y326L amino acid changeshave minimal effect on Yarrowia DGAT2 activity when assayed in yeast andthese two mutants were chosen for expression in soy somatic embryos.

Constructing Soy Expression Vectors Containing Mutant Yarrowia DGAT2s

The NotI fragment of pKR1254 (SEQ ID NO:78), pKR1254_Y326F (SEQ IDNO:81) or pKR1254_Y326L (SEQ ID NO:86), containing wild-type or mutantforms of Yarrowia DGAT2, were cloned into the NotI fragment of pKR72(SEQ ID NO:26; Example 4) to produce pKR1256 (SEQ ID NO:98), pKR1277(SEQ ID NO:99) or pKR1278 (SEQ ID NO:100), respectively.

Determining Oil Concentrations of Soy Somatic Embryos Expressing MutantYarrowia DGAT2s

Soybean embryogenic suspension culture (cv. Jack) was transformed witheither pKR1256 (SEQ ID NO:98), comprising wild-type Yarrowia DGAT2 andhaving experiment number MSE2228, pKR1277 (SEQ ID NO:99), comprisingYarrowia

DGAT2_Y326F and having experiment number MSE2229, or pKR1278 (SEQ IDNO:100), comprising Yarrowia DGAT2_Y326L and having experiment numberMSE2230. Events were selected and somatic embryos matured in SHaM asdescribed in Example 5. Oil concentrations were determined for eachevent using the NMR and described herein and fatty acid profiles weredetermined by GC exactly as described in herein and results for oilconcentrations and oleic acid content (% of total FAME) are shown inTable 32.

TABLE 32 Oil concentrations for somatic soy embryos transformed withpKR1256, pKR1277 or pKR1278 MSE2228-pKR1256 MSE2229-pKR1277MSE2230-pKR1278 (wt DGAT2) (Y326 F) (Y326 L) oleic acid oleic acid oleicacid FAME (% total FAME (% total FAME (% total Event # (% DCW) FAME)Event # (% DCW) FAME Event # (% DCW) FAME) 2228-9 14.3 44.4 2229-6 15.143.5 2230-4 11.6 35.1 2228-20 12.6 34.3 2229-20 14.4 44.3 2230-21 10.122.3 2228-3 12.4 39.3 2229-13 14.4 38.1 2230-15 9.8 22.4 2228-14 12.336.8 2229-21 14.0 36.5 2230-8 9.7 34.9 2228-2 11.7 36.8 2229-16 13.236.3 2230-29 9.6 33.0 2228-15 10.9 35.2 2229-24 13.1 39.9 2230-27 9.435.6 2228-5 10.1 23.3 2229-23 12.3 42.7 2230-3 9.4 36.7 2228-17 10.023.5 2229-3 12.2 41.3 2230-19 9.0 34.7 2228-24 10.0 27.6 2229-25 12.040.4 2230-6 8.6 23.6 2228-6 9.5 25.2 2229-31 11.9 39.0 2230-24 8.5 23.32228-18 9.5 32.4 2229-18 11.6 40.8 2230-18 8.4 35.8 2228-21 9.0 32.92229-5 11.4 38.7 2230-22 7.3 23.3 2228-12 8.7 22.7 2229-12 11.3 36.72230-2 7.0 26.7 2228-10 8.4 24.3 2229-10 11.3 35.6 2230-14 6.9 25.92228-22 7.5 29.9 2229-27 10.9 27.8 2230-9 6.9 22.9 2228-8 6.8 21.42229-30 10.7 39.0 2230-25 6.8 27.3 2228-19 6.5 27.3 2229-15 10.4 36.32230-7 6.8 37.5 2228-23 6.4 26.5 2229-8 9.7 39.5 2230-5 6.5 25.6 2228-76.1 22.2 2229-9 9.4 37.4 2230-10 6.4 31.1 2228-13 6.1 24.0 2229-22 9.222.3 2230-30 6.3 24.7 2228-11 5.2 24.3 2229-7 8.7 23.0 2230-26 6.3 23.42228-4 5.1 18.5 2229-26 8.4 31.2 2230-17 6.3 25.2 2228-16 4.2 19.32229-17 8.3 38.2 2230-20 6.3 19.5 2228-1 3.2 23.1 2229-28 7.9 27.22230-13 5.8 22.3 average 8.6 28.1 2229-29 7.8 25.7 2230-12 5.7 21.72229-1 7.4 32.8 2230-23 5.6 25.1 2229-2 5.9 18.6 2230-11 5.5 20.72229-11 4.6 18.6 2230-1 5.4 26.3 2229-19 4.1 22.6 2230-16 5.3 24.02229-4 4.0 21.2 2230-28 4.6 20.4 2229-14 3.1 39.8 2230-31 4.3 18.3average 8.8 32.2 average 6.5 25.4

In soy somatic embryos, a variant of the YL DGAT2 protein carrying theY326F mutation increases oil concentrations and shifts the fatty acidprofile of the oil at least to the same extent as the wild-type YarrowiaDGAT2.

Example 17 Expression Optimized DGAT Genes

Sequences encoding YL DGAT1 and YL DGAT2 genes that are optimized forexpression in soybean plants are set forth as SEQ ID NO:64 and SEQ IDNO:66. The design of these sequences is described in Example 9. DNAmolecules with this DNA sequence flanked by Not I restriction sites weresynthesized by DNA 2.0 (California, USA). Plasmid DNA with thesynthesized genes was digested with Not I. Not I restriction fragmentswith the DGAT genes were ligated to Not linearized, dephosphorylated DNAof KS332, which is described in Example 5. The resulting DNA constructsin which expression of expression-optimized variants of yarrowia DGAT1or yarrowia DGAT2 genes are under the control of the betaconglycininpromoter are henceforth referred to as KS392 and KS393. Their sequenceis set forth as SEQ ID NO:101 and SEQ ID NO:102. Moreover plasmid KS391was constructed. To this end DNA of KS349 was digested with NotI andNcoI. Ends of the of the resulting DGAT1 restriction fragment werecompletely filled-in and ligated to NotI linearized and filled in DNA ofKS332. The resulting plasmid construct is henceforth referred to asKS391. In this construct the native YL DGAT1 sequences is under thecontrol of the betaconglycinin promoter. The sequence of KS391 is setforth as SEQ ID NO:103. Transgenic soybean somatic embryos wereregenerated as after particle bombardment with plasmid DNA of KS391,KS392, KS 362 and KS393 as described above (Example 5). Oil content ofsomatic embryos was measured using NMR. Briefly lyophilized embryotissue was pulverized in genogrnder vial as described previously(Example 4). 20-200 mg of tissue powder were transferred to NMR tubes.Oil content of the somatic embryo tissue powder was calculated from theNMR signal as described in Example 4.

TABLE 33 Oil concentrations for somatic soy embryos transformed withpKS392 and pKS391 Construct KS392 Construct KS391 SAMPLE ID % oil SAMPLEID % oil 1 2196.1.08 15.7 1 2195.3.15 12.8 2 2196.3.03 13.9 2 2195.5.0211.5 3 2196.1.05 13.7 3 2195.3.01 11.2 4 2196.1.15 13.4 4 2195.3.03 11.05 2196.1.02 12.2 5 2195.3.02 10.6 6 2196.3.07 12.1 6 2195.4.06 10.4 72196.3.04 12.1 7 2195.3.08 10.2 8 2196.3.05 11.6 8 2195.2.01 10.1 92196.1.06 11.4 9 2195.3.04 9.3 10 2196.1.07 11.4 10 2195.3.05 9.2 112196.1.03 10.4 11 2195.5.01 9.1 12 2196.1.14 10.2 12 2195.2.04 8.8 132196.1.12 9.4 13 2195.3.14 8.6 14 2196.3.08 9.1 14 2195.3.10 7.8 152196.3.09 8.1 15 2195.4.01 7.7 16 2196.4.02 8.1 16 2195.5.03 6.5 172196.1.09 7.8 17 2195.4.02 6.4 18 2196.2.01 7.6 18 2195.3.09 6.3 192196.5.01 7.4 19 2195.4.07 6.2 20 2196.3.01 7.4 20 2195.3.06 6.1 212196.1.04 7.3 21 2195.3.13 6.0 22 2196.2.02 7.3 22 2195.5.05 5.9 232196.3.02 6.9 23 2195.4.03 5.6 24 2196.1.10 6.5 24 2195.5.04 5.5 252196.4.03 6.5 25 2196.1.01 5.3 26 2196.1.11 6.0 26 2195.4.04 5.3 272196.4.01 5.7 27 2195.3.11 5.2 28 2196.3.06 5.4 28 2195.2.03 5.0 292196.5.02 5.3 29 2195.4.05 4.9 30 2196.1.13 3.5 30 2195.3.07 4.4 312195.2.02 4.3 AVERAGE AVERAGE % OIL 9.1 % OIL 7.6

Table 33 compares the oil content of 30 and 31 events generated withKS392 and KS 391, respectively. Average oil content of all eventsgenerated with KS392 was 9.1% whereas oil content of all eventsgenerated with KS391 was 7.6%. More over the highest oil contentobserved with KS392 was 15.7% compared to 12.8% for KS391. Applicantshave demonstrated that expression optimization of YL DGAT1 leads toincreased oil content in developing soybean embryos when compared to thenative YL DGAT1 gene.

TABLE 34 Oil concentrations for somatic soy embryos transformed withpKS393 and pKS362 Construct KS393 Construct KS362 SAMPLE ID % oil SAMPLEID % oil  1 2207.5.05 12.3 1 2208.2.08 11.6  2 2207.5.08 12.2 22208.5.04 11.5  3 2207.5.06 11.7 3 2208.2.04 11.5  4 2207.5.03 10.8 42208.2.10 11.2  5 2207.5.01 10.6 5 2208.2.09 10.3  6 2207.5.04 10.3 62208.2.02 10.2  7 2207.4.04 10.3 7 2208.5.02 10.0  8 2207.3.09 9.5 82208.3.10 9.8  9 2207.5.07 9.3 9 2208.3.12 9.8 10 2207.4.01 8.8 102208.3.06 9.5 11 2207.4.02 8.7 11 2208.3.04 8.8 12 2207.3.06 8.0 122208.5.05 8.6 13 2207.3.04 7.9 13 2208.2.07 8.1 14 2207.4.06 7.8 142208.2.03 8.0 15 2207.4.03 7.7 15 2208.3.03 8.0 16 2207.4.08 7.4 162208.5.01 8.0 17 2207.3.14 7.1 17 2208.2.06 7.8 18 2207.4.05 7.0 182208.2.11 7.4 19 2207.3.12 7.0 19 2208.5.07 7.3 20 2207.3.01 6.9 202208.3.14 7.2 21 2207.5.02 6.8 21 2208.3.02 7.0 22 2207.3.02 6.7 222208.3.08 6.4 23 2207.3.07 6.7 23 2208.3.07 6.1 24 2207.3.03 6.6 242208.2.12 6.1 25 2207.3.13 6.5 25 2208.3.09 6.0 26 2207.3.11 6.3 262208.3.11 6.0 27 2207.3.05 5.8 27 2208.2.01 5.8 28 2207.3.10 5.8 282208.2.05 5.7 29 2207.4.07 5.5 29 2208.3.01 5.4 30 2207.3.08 5.4 302208.5.03 5.1 31 2207.4.09 5.4 31 2208.3.13 4.7 32 2208.5.06 4.1 332208.3.05 3.2 AVERAGE AVERAGE % OIL 8.0 % OIL 7.8

Table 34 compares the oil content 31 of and 33 events generated withKS393 and KS 362. Average oil content of all events generated with KS393was 8.0% whereas oil content of all events generated with KS393 was7.8%. More over the highest oil content observed with KS393 was 12.3%compared to 11.6% for KS362. Applicants have demonstrated thatexpression optimization of YL DGAT2 leads a very small increase in oilcontent in developing soybean embryos when compared to the native YLDGAT2 gene.

Example 18 Co-Expression of Either YL LPAAT1 or YL LPAAT2 with YL DGAT1and YL DGAT2 in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1242, comprising Yarrowia lyso-phosphatidic acid acyltransferaseprotein homolog 1 (YL LPAAT1) and pKR1243, comprising Yarrowialyso-phosphatidic acid acyltransferase protein homolog 2 (YL LPAAT2),and co-expression of these with pKR1236, comprising Yarrowia DGAT1 andDGAT2, in somatic embryos. Vector pKR1236 alone was also included as acontrol.

Construction of pKR1242 Comprising YL LPAAT1

The NcoI/BsiWI fragment of pFBAIn-YLPAT1 (SEQ ID NO:104), containing theYL LPAAT1, was cloned into the NcoI/BsiWI fragment of pKR908 (US Pat.Pub. 20080095915), to produce pKR1239 (SEQ ID NO:105) which generatesNotI sites flanking the YL LPAAT1 gene.

A starting vector pKR457 (SEQ ID NO:106), which was previously describedin PCT Publication No. WO 2005/047479 (the contents of which are herebyincorporated by reference), contains a NotI site flanked by the Kunitzsoybean Trypsin Inhibitor (KTi) promoter (Jofuku et al., Plant Cell1:1079-1093 (1989)) and the KTi 3′ termination region, the isolation ofwhich is described in U.S. Pat. No. 6,372,965, followed by the soyalbumin transcription terminator, which was previously described in PCTPublication No. WO 2004/071467.

The NotI fragment of pKR1239 (SEQ ID NO:105), containing YL LPAAT1, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1242(SEQ ID NO:107).

Construction of pKR1243 Comprising YL LPAAT2

The NcoI/BsiWI fragment of pFBAIn-YLPAAT2 (SEQ ID NO:108), containingthe YL LPAAT2, was cloned into the NcoI/BsiWI fragment of pKR908 toproduce pKR1240 (SEQ ID NO:109) which generates NotI sites flanking YLLPAAT2 gene. The NotI fragment of pKR1240 (SEQ ID NO:109), containing YLLPAAT2, was cloned into the NotI site of pKR457 (SEQ ID NO:106) toproduce pKR1243 (SEQ ID NO:110).

Co-Expression of Either YL LPAAT1 or YL LPAAT2 with YL DGAT1 and YLDGAT2

Vector pKR1242 (SEQ ID NO:107) and pKR1243 (SEQ ID NO:110) were digestedwith BsiWI and fragment purified as described herein (Example 5).Soybean embryogenic suspension culture (cv. Jack) was transformed withthe BsiWI fragment from pKR1242 (SEQ ID NO:107) and pKR1236 (SEQ IDNO:75) and having experiment number MSE2183 or with BsiWI fragment ofpKR1243 (SEQ ID NO:110) and pKR1236 (SEQ ID NO:75) and having experimentnumber MSE2184. Plasmid pKR1236 (SEQ ID NO:75) was also transformedalone in a similar way for a control and has experiment number MSE2185.Events were selected and somatic embryos matured in SHaM as described inExample 5. Oil concentrations and fatty acid profiles were determined asdescribed in Example 4 for MSE2183, MS2184 and MSE2185 and results foreach experiment are shown in Table 35, Table 36 and Table 37,respectively.

TABLE 35 Oil concentrations and fatty acid profiles for events fromMSE2183 MSE2183 (YL LPAAT1 + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2183-8 12.6 5.3 34.3 40.0 7.9 11.8 2183-26 11.8 5.4 42.234.2 6.4 11.2 2183-24 12.0 6.1 35.2 38.3 8.3 11.0 2183-23 12.6 6.5 33.738.6 8.7 10.9 2183-1 12.7 6.0 38.4 35.0 8.0 10.8 2183-15 13.2 6.0 32.039.1 9.7 10.5 2183-12 13.5 5.3 35.7 36.5 9.0 9.9 2183-27 13.6 5.5 29.740.5 10.7 9.8 2183-11 12.4 5.3 33.0 38.3 11.0 8.8 2183-10 14.5 6.0 29.438.7 11.4 8.0 2183-28 13.9 6.2 29.6 40.0 10.3 7.8 2183-17 13.8 6.3 32.337.0 10.7 7.7 2183-33 16.3 6.2 23.4 42.2 11.9 7.6 2183-7 14.2 6.3 31.237.2 11.2 7.1 2183-19 13.2 5.3 35.1 36.2 10.2 6.9 2183-30 15.3 5.6 28.438.5 12.1 6.9 2183-21 14.3 6.1 33.1 35.6 10.9 6.6 2183-2 12.8 4.5 29.341.4 12.1 6.6 2183-13 12.8 5.2 34.1 36.9 11.0 6.4 2183-20 16.6 5.3 22.442.5 13.2 6.4 2183-3 16.6 5.4 23.4 40.3 14.3 6.3 2183-5 14.2 5.8 31.436.6 11.9 6.2 2183-22 15.1 5.2 24.6 40.2 14.9 6.2 2183-6 17.7 5.0 20.741.3 15.3 6.0 2183-18 15.9 5.0 23.0 41.5 14.6 5.7 2183-25 17.4 4.1 15.445.6 17.5 5.5 2183-29 17.3 5.9 22.7 39.0 15.2 4.3 2183-9 17.9 5.7 21.937.6 16.9 4.3 2183-4 18.4 4.2 15.1 42.8 19.5 3.4 2183-32 20.1 3.8 15.039.4 21.7 3.4 Avg. 14.8 5.5 28.5 39.0 12.2 7.5

TABLE 36 Oil concentrations and fatty acid profiles for events fromMSE2184 MSE2184 (YL LPAAT2 + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2184-27 12.5 5.9 32.4 40.8 8.3 11.0 2184-24 13.1 6.9 34.137.1 8.8 9.5 2184-30 12.6 5.2 32.4 40.2 9.6 9.1 2184-20 14.3 5.6 26.842.4 10.9 9.1 2184-13 12.1 5.4 31.7 40.8 10.0 9.0 2184-18 14.5 5.3 28.441.8 10.1 8.9 2184-12 12.5 5.8 36.3 36.8 8.6 8.7 2184-16 14.1 5.4 29.540.8 10.1 8.5 2184-1 13.1 6.4 33.4 37.5 9.5 8.1 2184-8 18.7 5.2 18.744.1 13.3 8.1 2184-23 13.4 5.8 33.6 37.8 9.3 8.0 2184-4 14.5 5.1 30.939.5 10.0 7.9 2184-14 14.5 5.5 34.4 35.3 10.3 7.9 2184-29 12.7 5.7 31.440.0 10.1 7.9 2184-3 13.4 5.7 33.5 37.3 10.1 7.7 2184-9 14.2 4.8 29.239.5 12.2 7.4 2184-26 16.0 5.2 19.4 45.6 13.8 7.1 2184-2 15.8 4.8 24.742.7 12.0 6.1 2184-17 13.2 5.4 27.3 41.8 12.3 6.1 2184-31 17.0 4.9 18.043.5 16.6 5.6 2184-6 17.8 5.2 20.9 41.1 15.0 5.5 2184-25 17.1 6.0 23.638.7 14.5 5.2 2184-22 15.9 4.6 21.4 42.1 16.0 5.0 2184-21 16.6 4.6 16.844.8 17.2 4.9 2184-28 16.2 4.6 20.4 42.3 16.5 4.8 2184-5 15.5 4.6 16.843.9 19.2 4.7 2184-19 16.7 5.5 22.6 39.9 15.2 4.3 2184-7 16.0 4.0 13.639.9 26.5 3.6 Avg. 14.8 5.3 26.5 40.6 12.7 7.1

TABLE 37 Oil concentrations and fatty acid profiles for events fromMSE2185 MSE2185 (YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:1 18:2 18:3Oil 2185-17 12.6 6.4 32.8 39.6 8.6 11.8 2185-26 11.2 6.4 36.3 38.5 7.611.7 2185-18 11.8 6.5 38.8 35.7 7.1 11.6 2185-25 12.1 6.3 34.0 39.0 8.611.3 2185-15 12.2 5.9 36.8 37.6 7.6 11.1 2185-23 12.7 6.1 30.2 41.7 9.310.7 2185-21 11.8 6.5 35.3 38.1 8.3 10.5 2185-20 12.3 7.1 34.1 37.8 8.710.2 2185-31 13.6 6.4 34.4 37.0 8.5 10.1 2185-27 12.5 5.9 32.0 40.0 9.610.1 2185-14 13.2 5.9 31.7 39.7 9.5 9.5 2185-2 14.7 5.6 27.3 41.7 10.89.4 2185-6 12.4 6.2 35.3 37.7 8.3 9.2 2185-11 13.4 6.0 30.9 39.0 10.69.1 2185-19 13.5 6.1 30.2 39.1 11.0 9.1 2185-16 13.2 5.8 30.4 39.0 11.69.0 2185-1 15.6 5.0 24.5 42.9 11.9 8.6 2185-28 16.6 5.1 21.7 44.3 12.38.5 2185-3 13.9 6.0 30.4 39.2 10.5 8.4 2185-13 14.4 6.1 27.4 41.3 10.88.2 2185-5 14.1 5.5 31.4 38.6 10.4 7.4 2185-24 15.3 6.9 26.0 38.8 12.97.2 2185-4 18.1 5.4 22.4 39.3 14.8 6.3 2185-7 16.6 6.3 15.1 43.6 18.45.5 2185-29 15.9 6.0 24.3 39.4 14.4 5.1 2185-9 17.4 5.9 20.0 40.9 15.84.8 2185-30 15.6 5.0 17.5 41.2 20.8 4.3 Avg. 14.0 6.0 29.3 39.6 11.1 8.9

Example 19 Co-Expression of Either YL LPAAT3 or YL PDAT with YL DGAT1and YL DGAT2 in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1244, comprising Yarrowia lyso-phosphatidic acid acyltransferaseprotein homolog 3 (YL LPAAT3) and pKR1246, comprising Yarrowiaphospholipid:diacylglyceride acyltransferase (YL PDAT), andco-expression of these with pKR1236, comprising Yarrowia DGAT1 andDGAT2, in somatic embryos. Vector pKR1236 alone was also included as acontrol.

Construction of pKR1244 Comprising YL LPAAT3

The NcoI/BsiWI fragment of pFBAIn-YLPAT3 (SEQ ID NO:111), containing theYL LPAAT3, was cloned into the NcoI/BsiWI fragment of pKR908 (US Pat.Pub. 20080095915), to produce pKR1241 (SEQ ID NO:112) which generatesNotI sites flanking the YL LPAAT3 gene.

The NotI fragment of pKR1241 (SEQ ID NO:112), containing YL LPAAT3, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1244(SEQ ID NO:113).

Construction of pKR1246 Comprising YL PDAT

A starting vector pY27-PDAT (SEQ ID NO:114), which is described in U.S.Pat. No. 7,267,976, contains the YL PDAT flanked by NotI sites.

The NotI fragment of pY27-PDAT, containing YL PDAT, was cloned into theNotI site of pKR457 (SEQ ID NO:106) to produce pKR1246 (SEQ ID NO:115).

Co-Expression of Either YL LPAAT3 or YL PDAT with YL DGAT1 and YL DGAT2

Vector pKR1244 (SEQ ID NO:112) or pKR1246 (SEQ ID NO:114) were digestedwith BsiWI or SbfI/SmaI, respectively and fragment was purified asdescribed herein (Example 5). Soybean embryogenic suspension culture(cv. Jack) was transformed with BsiWI fragment from pKR1244 (SEQ IDNO:113) and pKR1236 (SEQ ID NO:75) and having experiment number MSE2189or with the SbfI/SmaI fragment of pKR1246 (SEQ ID NO:115) and pKR1236(SEQ ID NO:75) and having experiment number MSE2190. Plasmid pKR1236(SEQ ID NO:75) was also transformed alone in a similar way for a controland has experiment number MSE2191. Events were selected and somaticembryos matured in SHaM as described in Example 5. Oil concentrationsand fatty acid profiles were determined as described in Example 4 forMSE2189, MS2190 and MSE2191 and results for each experiment are shown inTable 38, Table 39 and Table 40, respectively.

TABLE 38 Oil concentrations and fatty acid profiles for events fromMSE2189 MSE2189 (YL LPAAT3 + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2189-31 14.1 6.2 32.7 38.6 8.4 9.7 2189-22 14.3 6.5 32.637.5 9.1 9.6 2189-30 12.7 7.0 39.0 33.4 7.8 9.4 2189-2 13.5 6.0 37.034.8 8.8 9.4 2189-27 12.5 6.0 36.5 36.3 8.7 8.6 2189-19 14.1 5.4 28.240.8 11.5 8.2 2189-4 15.2 5.6 28.3 37.2 13.7 8.0 2189-7 14.9 6.9 28.638.3 11.3 7.9 2189-21 14.4 6.5 31.8 37.3 10.0 7.5 2189-13 15.4 7.0 34.134.7 8.8 7.2 2189-12 13.8 5.7 32.2 36.2 12.0 6.9 2189-20 16.7 5.1 19.244.6 14.3 6.3 2189-5 15.6 6.2 31.0 35.9 11.3 6.2 2189-25 15.9 6.6 24.339.0 14.2 6.0 2189-23 15.9 5.4 23.0 40.6 15.1 5.5 2189-28 20.7 5.2 16.843.2 14.1 5.3 2189-29 16.7 5.2 21.5 40.6 16.0 5.0 2189-1 18.8 4.8 16.541.0 18.9 4.9 2189-15 20.2 6.4 25.2 36.8 11.4 4.6 2189-8 17.9 4.8 17.141.0 19.1 4.1 2189-9 18.0 6.3 17.4 40.1 18.1 4.0 2189-24 16.6 5.4 19.940.5 17.6 4.0 2189-18 17.6 5.2 20.2 39.9 17.0 4.0 2189-3 17.7 5.2 19.139.4 18.6 3.8 Avg. 16.0 5.9 26.3 38.7 13.2 6.5

TABLE 39 Oil concentrations and fatty acid profiles for events fromMSE2190 MSE2190 (YL PDAT + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2190-13 14.4 6.2 32.9 38.1 8.5 11.1 2190-6 13.6 7.1 35.036.3 8.1 10.3 2190-27 12.5 5.6 44.4 30.5 6.9 9.8 2190-18 12.9 6.0 36.835.2 9.1 9.0 2190-8 14.1 6.1 31.7 36.7 11.5 8.1 2190-7 13.9 5.8 31.637.7 11.0 7.7 2190-2 15.0 5.7 20.1 44.9 14.3 7.2 2190-20 14.1 6.5 30.138.0 11.3 7.1 2190-30 17.5 6.9 24.3 38.2 13.1 7.1 2190-26 16.1 5.6 25.538.8 13.9 7.0 2190-12 15.2 6.7 31.1 34.8 12.2 6.8 2190-1 14.7 5.0 25.740.7 13.9 6.3 2190-28 16.6 5.3 19.9 43.2 15.0 6.3 2190-16 14.0 8.6 27.438.6 11.5 6.1 2190-5 16.2 5.8 24.2 40.5 13.3 6.1 2190-14 16.2 5.6 21.441.4 15.4 6.1 2190-3 17.3 5.5 20.9 39.5 16.8 6.1 2190-29 16.5 5.7 24.940.1 12.9 6.0 2190-24 16.5 5.4 21.9 40.3 15.9 5.9 2190-4 18.7 5.9 20.441.4 13.5 5.8 2190-23 17.6 4.9 15.8 43.7 18.0 5.5 2190-9 17.5 5.1 17.544.3 15.6 5.3 2190-21 16.6 5.8 20.7 42.0 14.9 5.2 2190-19 16.6 4.9 17.440.9 20.2 5.0 2190-10 17.4 6.4 22.9 38.9 14.4 4.7 2190-17 17.1 6.3 23.538.4 14.6 4.7 2190-15 16.7 5.3 19.9 40.2 17.9 4.6 2190-11 16.1 5.0 18.839.6 20.5 4.0 Avg. 15.8 5.9 25.2 39.4 13.7 6.6

TABLE 40 Oil concentrations and fatty acid profiles for events fromMSE2191 MSE2191 (YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:1 18:2 18:3Oil 2191-17 12.6 6.6 37.6 36.0 7.1 11.2 2191-30 13.1 5.8 38.2 35.4 7.510.6 2191-29 12.7 4.5 33.9 39.6 9.2 10.6 2191-2 12.6 6.3 35.5 36.7 8.99.9 2191-26 13.1 6.6 35.5 36.5 8.4 9.8 2191-8 13.0 6.5 35.6 36.5 8.4 9.62191-1 13.1 5.5 36.9 36.4 8.1 9.4 2191-24 12.8 4.6 34.1 39.4 9.1 9.32191-5 13.0 6.2 36.3 36.0 8.5 9.0 2191-20 13.6 6.9 34.1 35.5 9.9 8.82191-28 13.1 6.6 33.9 36.2 10.2 8.7 2191-23 12.7 5.6 36.7 36.0 9.0 8.72191-12 13.0 5.4 36.1 35.4 10.0 8.6 2191-9 14.5 7.1 29.3 37.4 11.7 8.22191-15 14.4 5.8 32.2 36.6 10.9 7.0 2191-7 15.9 5.7 29.5 38.1 10.9 6.12191-27 16.9 6.0 22.5 39.6 15.0 5.7 2191-14 16.0 5.5 24.4 39.1 15.0 5.72191-25 18.2 5.8 22.1 37.6 16.3 5.3 2191-19 16.8 6.0 22.3 39.9 15.0 5.12191-21 15.8 4.5 24.0 39.6 16.2 4.8 2191-6 17.4 5.5 22.0 38.3 16.9 4.42191-3 17.6 4.8 20.0 40.6 17.0 4.4 2191-18 19.1 6.3 21.1 39.3 14.3 4.22191-16 17.1 4.8 17.0 40.5 20.6 4.0 2191-10 17.5 4.6 21.0 38.3 18.6 3.72191-13 18.4 4.6 16.2 40.9 20.0 3.3 2191-22 17.4 4.6 15.3 40.8 21.9 3.1Avg. 15.1 5.7 28.7 37.9 12.7 7.1

Example 20 Co-Expression of Either YL ACBP or YL CPT with YL DGAT1 andYL DGAT2 in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1250, comprising Yarrowia acyl-CoA binding protein homolog (YL ACBP)and pKR1251, comprising Yarrowia choline phosphotransferase homolog (YLCPT), and co-expression of these with pKR1236, comprising Yarrowia DGAT1and DGAT2, in somatic embryos. Vector pKR1236 alone was also included asa control.

Construction of pKR1250 comprising YL ACBP

The NcoI/BsiWI fragment of pYAT-ACBP (SEQ ID NO:116), containing the YLACBP, was cloned into the NcoI/BsiWI fragment of pKR908 (US Pat. Pub.20080095915), to produce pKR1245 (SEQ ID NO:117) which generates NotIsites flanking the YL ACBP gene.

The NotI fragment of pKR1245 (SEQ ID NO:116), containing YL ACBP, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1250(SEQ ID NO:118).

Construction of pKR1251 comprising YL CPT

The NcoI/BsiWI fragment of pFBAIn-YCPT1 (SEQ ID NO:119), containing theYL CPT, was cloned into the NcoI/BsiWI fragment of pKR908 to producepKR1247 (SEQ ID NO:120) which generates NotI sites flanking YL CPT gene.

The NotI fragment of pKR1247 (SEQ ID NO:120), containing YL CPT, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1251(SEQ ID NO:121).

Co-Expression of Either YL ACBP or YL CPT with YL DGAT1 and YL DGAT2

Vector pKR1250 (SEQ ID NO:118) and pKR1251 (SEQ ID NO:121) were digestedwith BsiWI and fragment purified as described in Example 5. Soybeanembryogenic suspension culture (cv. Jack) was transformed with the BsiWIfragment from pKR1250 (SEQ ID NO:118) and pKR1236 (SEQ ID NO:75) andhaving experiment number MSE2192 or with the BsiWI fragment of pKR1251(SEQ ID NO:121) and pKR1236 (SEQ ID NO:75) and having experiment numberMSE2193. Plasmid pKR1236 (SEQ ID NO:75) was also transformed alone in asimilar way for a control and has experiment number MSE2194. Events wereselected and somatic embryos matured in SHaM as described in Example 5.Oil concentrations and fatty acid profiles were determined as describedin Example 4 for MSE2192, MS2193 and MSE2194 and results for eachexperiment are shown in Table 41, Table 42 and Table 43, respectively.

TABLE 41 Oil concentrations and fatty acid profiles for events fromMSE2192 MSE2192 (YL ACBP + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2192-2 13.1 6.2 33.5 38.6 8.5 13.8 2192-5 12.0 4.7 41.534.2 7.4 12.8 2192-24 13.1 5.9 34.5 38.1 8.4 12.3 2192-27 12.5 6.5 34.737.6 8.7 11.5 2192-30 13.1 6.2 34.2 37.1 9.4 11.5 2192-1 13.9 5.5 26.043.6 11.0 10.6 2192-25 14.0 6.6 28.2 38.7 12.5 10.2 2192-17 13.2 7.031.3 38.1 10.4 10.1 2192-29 14.1 6.5 30.0 38.6 10.8 10.1 2192-13 15.56.5 31.4 36.2 10.5 9.9 2192-20 12.9 5.6 31.7 38.4 11.4 9.8 2192-14 14.26.5 28.9 38.7 11.8 9.6 2192-15 13.8 5.9 31.5 39.0 9.7 9.5 2192-9 14.16.5 29.4 38.7 11.3 8.7 2192-12 13.9 6.2 29.6 37.4 12.9 8.4 2192-11 14.26.3 28.5 38.6 12.4 8.4 2192-6 13.9 6.2 29.2 38.2 12.5 8.0 2192-19 16.25.8 22.5 41.4 14.0 7.8 2192-23 13.9 5.8 28.5 39.0 12.7 7.5 2192-4 14.56.1 27.9 39.1 12.5 7.4 2192-31 15.9 6.0 21.2 42.0 14.9 7.3 2192-10 16.76.0 19.1 43.1 15.1 7.1 2192-28 15.5 5.0 24.0 40.2 15.4 6.5 2192-22 16.25.6 23.2 38.8 16.2 6.2 2192-26 17.2 5.3 18.0 41.1 18.4 5.8 2192-8 17.94.9 16.3 43.0 17.9 5.4 2192-16 17.7 5.3 15.1 41.8 20.1 4.8 2192-18 16.34.7 16.1 41.0 21.8 4.4 2192-3 17.0 4.6 16.0 40.3 22.1 4.1 Avg. 14.7 5.927.0 39.3 13.1 8.6

TABLE 42 Oil concentrations and fatty acid profiles for events fromMSE2193 MSE2193 (YL CPT + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2193-3 12.6 7.1 36.9 35.7 7.7 12.4 2193-22 13.2 7.4 35.634.4 9.4 12.2 2193-16 13.4 7.9 36.5 33.0 9.2 10.9 2193-10 13.9 6.0 32.238.1 9.8 10.1 2193-6 13.8 6.9 31.7 37.5 10.1 10.0 2193-14 13.5 6.7 30.539.3 10.0 9.9 2193-20 14.4 6.8 29.8 37.2 11.7 9.7 2193-8 13.4 7.0 33.835.9 9.9 9.2 2193-9 14.8 6.9 27.5 38.8 12.0 8.3 2193-18 15.5 6.4 26.138.4 13.5 7.2 2193-19 14.9 6.4 28.8 37.8 12.1 6.7 2193-17 16.9 6.4 24.037.7 15.0 6.5 2193-4 17.1 6.1 20.2 40.0 16.6 5.8 2193-11 16.2 5.7 22.238.8 17.1 5.5 2193-15 16.9 6.0 21.0 39.5 16.6 5.2 2193-7 17.1 6.3 21.239.1 16.3 5.1 2193-13 15.8 7.3 26.6 35.7 14.6 5.0 2193-21 17.2 6.5 21.738.0 16.6 4.7 2193-1 18.1 4.5 15.1 39.7 22.5 3.6 2193-2 19.7 5.1 14.940.8 19.6 3.4 Avg. 15.4 6.5 26.8 37.8 13.5 7.6

TABLE 43 Oil concentrations and fatty acid profiles for events fromMSE2194 MSE2194 (YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:1 18:2 18:3Oil 2194-16 12.2 5.8 41.6 33.2 7.2 12.0 2194-23 12.3 5.5 37.5 36.2 8.510.6 2194-5 15.1 5.6 30.6 36.6 12.2 10.0 2194-10 14.0 5.9 34.3 35.9 9.99.5 2194-15 13.6 5.7 32.1 36.8 11.8 9.4 2194-4 14.6 5.9 32.4 35.7 11.49.2 2194-20 13.1 6.1 33.1 37.0 10.7 8.8 2194-8 13.6 4.9 32.8 37.7 10.98.8 2194-11 11.8 5.3 38.3 33.4 11.2 8.7 2194-3 14.1 5.3 30.5 37.8 12.28.6 2194-13 13.8 5.5 31.0 37.7 11.9 8.5 2194-7 15.0 5.7 27.8 38.3 13.28.1 2194-22 14.5 4.9 28.7 38.5 13.4 7.4 2194-25 15.2 5.1 27.3 37.6 14.87.4 2194-21 15.1 5.4 25.9 40.1 13.5 7.3 2194-6 14.3 5.9 27.7 38.2 13.97.2 2194-27 13.9 5.2 30.3 37.5 13.1 7.2 2194-1 14.8 5.2 29.2 37.8 13.07.1 2194-17 14.2 4.6 27.9 37.2 16.0 6.8 2194-28 14.8 5.2 27.6 37.3 15.16.3 2194-14 16.0 5.0 21.2 41.6 16.2 6.2 2194-19 15.2 4.3 24.2 39.7 16.65.7 2194-2 15.8 4.8 17.0 45.1 17.4 5.5 2194-9 17.5 5.2 17.9 41.5 17.95.4 2194-12 16.8 4.9 18.3 39.5 20.5 4.5 2194-18 17.3 4.7 17.9 41.2 18.94.5 2194-24 16.5 4.0 15.5 43.1 20.9 4.4 2194-26 14.4 4.7 15.1 44.2 21.73.3 Avg. 14.6 5.2 27.6 38.5 14.1 7.4

The results in Tables 41-43 show that average oil concentrations arehigher when YL ACBP is co-expressed with YL DGAT1 and YL DGAT2 than whenYL DGAT1 and YL DGAT2 are expressed alone.

Example 21 Co-Expression of YL GPAT with YL DGAT1 and YL DGAT2 inSoybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1257, comprising Yarrowia glycerolphosphate acyltransferase homolog(YL GPAT) and co-expression with pKR1236, comprising Yarrowia DGAT1 andDGAT2, in somatic embryos. Vector pKR1236 alone was also included as acontrol. Construction of pKR1257 comprising YL GPAT

The nucleotide and amino acid sequences for YL GPAT are set forth in SEQID NO:122 and SEQ ID NO:123, respectively. YL GPAT (SEQ ID NO:122) wasamplified from Yarrowia genomic DNA as described in Example 2 witholigonucleotide primers YIGPAT-5 (SEQ ID NO:124) and YIGPAT-3 (SEQ IDNO:125), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce PCRblunt-YIGPAT (SEQID NO:126).

The NotI fragment of PCRblunt-YIGPAT (SEQ ID NO:126), containing YLGPAT, was cloned into the NotI site of pKR457 (SEQ ID NO:106) to producepKR1257 (SEQ ID NO:127).

Co-Expression of YL GPAT with YL DGAT1 and YL DGAT2

Vector pKR1257 (SEQ ID NO:127) was digested with BsiWI and the fragmentpurified as described in Example 5. Soybean embryogenic suspensionculture (cv. Jack) was transformed with the BsiWI fragment from pKR1257(SEQ ID NO:127) and pKR1236 (SEQ ID NO:75) and having experiment numberMSE2216. Plasmid pKR1236 (SEQ ID NO:75) was also transformed alone in asimilar way for a control and has experiment number MSE2217. Events wereselected and somatic embryos matured in SHaM as described in Example 5.Oil concentrations and fatty acid profiles were determined as describedin Example 4 for MSE2216 and MSE2217 and results for each experiment areshown in Table 44 and Table 45, respectively.

TABLE 44 Oil concentrations and fatty acid profiles for events fromMSE2216 MSE2216 (YL GPAT + YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:118:2 18:3 Oil 2216-4 12.9 6.6 29.0 42.8 8.8 12.6 2216-2 11.9 7.0 32.439.7 9.0 11.8 2216-30 11.5 7.6 35.3 37.0 8.6 11.5 2216-28 12.0 7.2 33.339.1 8.4 11.1 2216-17 13.6 6.1 32.7 39.0 8.5 11.0 2216-9 13.2 7.6 33.937.2 8.1 11.0 2216-24 11.6 7.5 35.8 37.1 7.9 11.0 2216-31 12.0 6.1 39.236.5 6.1 10.9 2216-11 12.7 6.7 30.9 39.5 10.2 9.9 2216-5 13.3 6.0 31.539.2 10.0 9.8 2216-12 13.1 6.8 31.4 38.8 10.0 9.3 2216-14 13.1 6.9 33.737.1 9.3 9.1 2216-13 13.5 6.5 28.4 40.2 11.4 8.3 2216-8 14.6 7.5 27.938.5 11.5 7.8 2216-22 14.2 7.8 27.6 38.4 11.9 7.8 2216-18 15.6 7.1 24.439.2 13.7 6.6 2216-20 15.9 7.0 22.7 41.7 12.7 6.5 2216-19 16.8 6.2 19.242.7 15.1 6.3 2216-27 15.3 6.5 23.1 40.5 14.6 6.2 2216-3 15.9 7.4 21.340.4 15.0 6.1 2216-16 16.7 7.0 21.1 40.2 15.1 5.9 2216-1 17.6 5.3 15.244.5 17.4 5.4 2216-26 17.1 6.1 18.0 42.4 16.4 5.3 2216-25 16.4 7.6 22.438.3 15.3 5.3 2216-15 17.3 7.2 21.3 40.4 13.8 5.2 2216-10 17.0 6.3 20.140.7 15.8 5.2 2216-29 16.2 7.5 20.6 37.5 18.2 5.0 2216-21 17.3 8.9 23.734.1 16.0 4.5 2216-6 17.2 5.7 16.8 43.5 16.8 4.4 2216-7 18.2 5.5 15.343.7 17.4 4.3 2216-23 18.4 6.0 17.3 39.8 18.5 3.3 Ava. 14.9 6.8 26.039.7 12.6 7.7

TABLE 45 Oil concentrations and fatty acid profiles for events fromMSE2217 MSE2217 (YL DGAT1 & YL DGAT2) % Event 16:0 18:0 18:1 18:2 18:3Oil 2217-11 11.6 6.1 33.8 40.4 8.1 13.8 2217-17 12.8 4.7 27.9 45.4 9.113.5 2217-25 10.6 6.5 37.4 38.4 7.1 13.2 2217-4 11.6 6.0 38.5 36.9 7.013.2 2217-27 11.2 4.7 42.6 34.5 7.0 13.0 2217-15 11.9 9.2 37.0 34.7 7.211.5 2217-21 12.3 7.4 33.0 38.2 9.1 11.0 2217-6 14.1 6.8 31.4 38.7 9.010.7 2217-14 13.5 7.5 32.8 37.6 8.6 10.7 2217-19 13.2 5.7 28.5 42.1 10.610.5 2217-26 11.6 7.3 36.5 36.8 7.7 10.3 2217-24 11.5 7.1 38.2 35.2 7.99.7 2217-5 12.4 6.8 31.9 38.9 10.0 9.7 2217-28 14.9 6.7 27.5 40.3 10.69.5 2217-12 12.8 6.7 33.0 37.6 9.9 9.4 2217-31 15.2 5.6 21.5 46.4 11.39.3 2217-18 13.0 6.7 33.4 37.2 9.7 9.0 2217-13 14.2 5.2 25.1 45.2 10.38.7 2217-22 13.6 6.7 30.2 38.2 11.2 8.5 2217-3 12.9 6.4 33.0 37.6 10.18.2 2217-20 13.9 7.3 29.0 37.6 12.1 7.9 2217-23 14.7 6.7 27.7 38.9 11.97.7 2217-2 14.1 6.9 28.4 39.0 11.7 7.5 2217-9 15.5 6.6 23.1 41.1 13.77.3 2217-7 16.5 5.6 20.1 42.8 15.1 7.3 2217-16 14.7 7.3 25.9 39.2 12.97.3 2217-10 15.4 6.6 22.1 42.2 13.7 7.3 2217-1 13.7 5.5 17.7 45.3 17.86.7 2217-30 16.1 7.0 22.8 40.4 13.7 6.7 2217-8 13.0 6.2 27.8 39.8 13.16.2 2217-29 15.4 6.2 21.0 42.0 15.5 5.2 Avg. 13.5 6.5 29.6 39.6 10.7 9.4

Example 22 Co-Expression of Either YL PAP1, YL PAP2 or YL PAP3 and YLDGAT1 YL DGAT2 in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1347, comprising Yarrowia phosphatidic acid phosphatase homolog 1 (YLPAP1), pKR1348, comprising Yarrowia phosphatidic acid phosphatasehomolog 3 (YL PAP3) and pKR1349, comprising Yarrowia phosphatidic acidphosphatase homolog 2 (YL PAP2), and co-expression of these withpKR1236, comprising Yarrowia DGAT1 and DGAT2, in somatic embryos. VectorpKR1236 alone was also included as a control.

Construction of pKR1347 comprising YL PAP1

The nucleotide and amino acid sequences for YL PAP1 are set forth in SEQID NO:128 and SEQ ID NO:129, respectively. YL PAP1 (SEQ ID NO:128) wasamplified from Yarrowia genomic DNA as described in Example 2 witholigonucleotide primers YIPAP1-5 (SEQ ID NO:130) and YIPAP1-3 (SEQ IDNO:131), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD33 (SEQ ID NO:132).

The NotI fragment of pHD33 (SEQ ID NO:132), containing YL PAP1, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1347(SEQ ID NO:133).

Construction of pKR1348 2 comprising YL PAP3

The nucleotide and amino acid sequences for YL PAP3 are set forth in SEQID NO:134 and SEQ ID NO:135, respectively. YL PAP3 (SEQ ID NO:134) wasamplified from Yarrowia genomic DNA as described in Example 2 witholigonucleotide primers YIPAP3-5 (SEQ ID NO:136) and YIPAP3-3 (SEQ IDNO:137), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD34 (SEQ ID NO:138).

The NotI fragment of pHD34 (SEQ ID NO:138), containing YL PAP3, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1348_2(SEQ ID NO:139).

Construction of pKR1349 comprising YL PAP2

The nucleotide and amino acid sequences for YL PAP2 are set forth in SEQID NO:140 and SEQ ID NO:141, respectively. YL PAP2 (SEQ ID NO:140) wasamplified from Yarrowia genomic DNA as described in Example 2 witholigonucleotide primers YIPAP2-5 (SEQ ID NO:142) and YIPAP2-3 (SEQ IDNO:143), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD35 (SEQ ID NO:144).

The NotI fragment of pHD35 (SEQ ID NO:144), containing YL PAP2, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1349(SEQ ID NO:145).

Co-Expression of Either YL PAP1, YL PAP2 or YL PAP3 with YL DGAT1 and YLDGAT2

Vector pKR1347 (SEQ ID NO:133), pKR1348_2 (SEQ ID NO:139) and pKR1349(SEQ ID NO:145) were digested with BsiWI and fragments purified asdescribed in Example 5. Soybean embryogenic suspension culture (cv.Jack) was transformed with the BsiWI fragment from pKR1347 (SEQ IDNO:133) and pKR1236 (SEQ ID NO:75) and having experiment number MSE2322or with the BsiWI fragment of pKR1348_2 (SEQ ID NO:139) and pKR1236 (SEQID NO:75) and having experiment number MSE2323 or with the BsiWIfragment of pKR1349 (SEQ ID NO:145) and pKR1236 (SEQ ID NO:75) andhaving experiment number MSE2324. Plasmid pKR1236 (SEQ ID NO:75) wasalso transformed alone in a similar way for a control and has experimentnumber MSE2325.

Example 23 Co-Expression of YL GPD and YL DGAT1 YL DGAT2 in SoybeanSomatic Embryos

The present example describes construction of soybean expression vectorpKR1369, comprising a Yarrowia glycerol phosphate dehydrogenase homolog(YL GPD), and co-expression with pKR1236, comprising Yarrowia DGAT1 andDGAT2, in somatic embryos. Vector pKR1236 alone was also included as acontrol.

Construction of pKR1369 Comprising YL GPD

The nucleotide and amino acid sequences for YL GPD are set forth in SEQID NO:146 and SEQ ID NO:147, respectively. YL GPD genomic sequencecontains an intron in the coding sequence and this was removed usingPCR. The 5′ end of YL GPD was amplified from Yarrowia genomic DNA asdescribed in Example 2 with oligonucleotides YIGPD-5 (SEQ ID NO:148) andYIGPD-3-2 (SEQ ID NO:149), using the Phusion™ High-Fidelity DNAPolymerase (Cat. No. F553S, Finnzymes Oy, Finland), following themanufacturer's protocol. The 3′ end of YL GPD was amplified in a similarway with oligonucleotides YIGPDintron-5-2 (SEQ ID NO:150) and YIGPD-3(SEQ ID NO:151). The two resulting PCR products were combined andre-amplified using YIGPD-5 (SEQ ID NO:148) and YIGPD-3 (SEQ ID NO:151).The resulting DNA fragment was cloned into the pCR-Blunt® cloning vectorusing the Zero Blunt® PCR Cloning Kit (Invitrogen Corporation),following the manufacturer's protocol, to produce pHD36 (SEQ ID NO:152).

An SbfI site was added to the 3′ end of the soy albumin terminator inpKR457 (SEQ ID NO:106) using PCR and the resulting plasmid, which isalmost identical to pKR457 (SEQ ID NO:106) is called pKR1273 (SEQ IDNO:153).

The NotI fragment of pHD36 (SEQ ID NO:152), containing YL GPD, wascloned into the NotI site of pKR457 (SEQ ID NO:106) to produce pKR1369(SEQ ID NO:154).

Co-Expression of YL GPD with YL DGAT1 and YL DGAT2

pKR1369 (SEQ ID NO:154) was digested with BsiWI and the fragmentpurified as described in Example 5. Soybean embryogenic suspensionculture (cv. Jack) was transformed with the BsiWI fragment from pKR1369(SEQ ID NO:154) and pKR1236 (SEQ ID NO:75) and having experiment numberMSE2357. Plasmid pKR1236 (SEQ ID NO:75) was also transformed alone in asimilar way for a control and has experiment number MSE2353.

Example 24 Co-Expression of Plant LPAAT-Like Sequences and YL DGAT1 YLDGAT2 in Soybean Somatic Embryos

The following example describes experiments that demonstrate thatco-expression of certain LPAAT-like proteins and YL DGAT proteins insoybean somatic embryos increases oil content and allows to achieve oillevels that exceed those observed when only YL DGAT genes are expressed.

WO2000049156, U.S. Pat. No. 7,235,714 describes EST clones from soybeanand the catalpa tree that show similarity to lysophosphatidic acidacyltransferase (LPAATs) enzymes. One LPAAT-like gene from soybean wasamplified from plasmid DNA of applicant's EST clone (s12.pk121.a19)using oligonucleotide primers. Nucleotide sequence of the EST clone andamino acid sequence of the corresponding protein are listed as SEQ IDNO:155 and SEQ ID NO:156 The sequences of the sense (forward) andantisense (reverse) oligonucleotide primers used in this reaction wereas follows:

(SEQ ID NO: 157) 5′-CACCATGAATGGCATTGGGAAACTCAAATC-3′ and(SEQ ID NO: 158) 5′-CATTATTTTTCCTCAAAGCGCCGCAACAC-3′.PCR amplification was achieved using TAQ polymerase, and plasmid DNA ofthe EST clone was used as the template. The product of this PCR reactionwas purified by agarose gel electrophoresis and subcloned intopENTR/D-TOPO (Invitrogen) as described in the manufacturer's protocol. A1149 bp fragment containing the entire open-reading frame of was excisedusing restriction enzymes AscI and NotI. Ends were completely filled inwith T4 polymerase (Invitrogen, USA) according to instructions of themanufacturer and ligated to NotI linearized, filled-in pKR561 vector.Recombinant clones were subjected to analysis by restriction enzymedigestion to identify ligation products in which the start codon was inproximity of the annexin promoter in pKR561 (sense orientation).Plasmids with this orientation are henceforth referred to as pKR561-SOYLPAAT-like (SEQ ID NO:159).

One LPAAT-like gene from the catalpa tree was amplified from plasmid DNAof applicants EST clone (ncs. pk0013.d2) using oligonucleotide primers.Nucleotide sequence of the EST clone and amino acid sequence of thecorresponding protein are listed as SEQ ID NO:160 and SEQ ID NO:161. Thesequences of the sense (forward) and antisense (reverse) oligonucleotideprimers used in this reaction were as follows:

(SEQ ID NO: 162) 5′-CACCATGAGCAAGCTAAAAACATCCAGC-3′ and (SEQ ID NO: 163)5′-CTTCAATCTATTTCTCTTCCAGGTG-3′.PCR amplification was achieved using TAQ polymerase, and plasmid DNA ofthe EST clone was used as the template. The product of this PCR reactionwas purified by agarose gel electrophoresis and subcloned intopENTR/D-TOPO (Invitrogen) as described in the manufacturer's protocol.An 1148 bp fragment containing the entire open-reading frame of wasexcised using restriction enzymes AscI and NotI. Ends were completelyfilled in with T4 polymerase (Invitrogen, USA) according to instructionsof the manufacturer and ligated to NotI linearized, filled-in pKR561vector. Recombinant clones were subjected to analysis by restrictionenzyme digestion to identify ligation products in which the start codonwas in proximity of the annexin promoter in pKR561 (sense orientation).Plasmids with this orientation are henceforth referred to aspKR561-CATALPA LPAAT-like (SEQ ID NO:164).

Vector pKR561 had previously been constructed as follows. Vector pKR268(SEQ ID NO:165), which was previously described in U.S. Pat. No.7,256,033 (the contents of which are hereby incorporated by reference),contains a NotI site flanked by the soybean annexin promoter (U.S. Pat.No. 7,129,089) and the BD30 3′ termination region (Ann/NotI/BD30cassette). Vector pKR145 (SEQ ID NO:166), which was previously describedin PCT Publication No. WO 2004/071467 (the contents of which are herebyincorporated by reference), contains the hygromycin B phosphotransferasegene [Gritz, L. and Davies, J. (1983) Gene 25:179-188], flanked by theT7 promoter and transcription terminator (T7prom/hpt/T7term cassette),and a bacterial origin of replication (ori) for selection andreplication in E. coli. In addition, pKR145 contains the hygromycin Bphosphotransferase gene, flanked by the 35S promoter [Odell et al.,(1985) Nature 313:810-812] and NOS3′ transcription terminator [Depickeret al., (1982) J. Mol. Appl. Genet. 1:561:570] (35S/hpt/NOS3′ cassette)for selection in soybean. The BsiWI fragment of pKR268, containing theAnn/NotI/BD30 cassette, was cloned into the BsiWI fragment of pKR145,containing the 35S/hpt/NOS3′ cassette), to produce pKR561 (SEQ IDNO:167).

For co-expression of YL DGAT1 YL DGAT2 and LPAAT-like sequences genes insoybean somatic embryos, soybean tissue was co-bombarded as describedherein (Example 5) with a mixture KS387 which is described below.Plasmid DNA of KS387 and pKR561-SOY LPAAT-like or KR561-CATALPALPAAT-like were combined in a 10:1 ratio and used for transformation ofsoybean somatic embryos as described below. In the resulting DNA mixtureKS387 provides expression cassettes for YL DGAT1 and YL DGAT2 whereasthe KR561 plasmids provide expression cassettes for LPAAT like genes andfor a gene generating hygromycin resistance comprised of CaMV 35Spromoter hygromycin phosphotransferase gene and nos terminator.

KS387 was constructed as follows. A 6818 bp restriction fragment wasexcised form KS364 (SEQ ID NO:63) by digestion with AscI. This DNAfragment contains expression cassettes for both YL DGAT genes. It wascompletely filled in with T4 polymerase and ligated to SalI-linearizedDNA of KS178 that had previously been treated with T4 polymerase togenerate blunt ends for ligation. The sequence of KS387 is set forth asSEQ ID NO:168.

Prior to this KS178 was constructed as follows. The 4.0 kb DNA fragmentcontaining the SAMS/ALS/ALS3′ cassette was excised from pZSL13LeuB (PCTPublication No. WO 04/071467) using the restriction enzymes PstI andSmaI, the ends were blunted with the large fragment of DNA polymerase I,and ligated to DNA of KS102 (PCT Publication No. WO 04/071467)linearized with the restriction enzyme BamHI, to give KS178 (SEQ IDNO:169). Prior to ligation the ends of the linearized KS102 vector wereblunted with the large fragment of DNA polymerase I.

Fatty acid profile and oil content of soybean somatic embryos generatedby co-transformation of YL DGATs with LPAAT-like genes from soy andcatalpa is shown in Tables 46 and Table 47, respectively. Fatty acidprofile and oil content of soybean somatic embryos generated bytransformation with YL DGATs only is shown in Tables 48. Average oilcontent of all events created is higher when YL DGATs are co-expressedwith LPAAT-like genes. Similarly, changes in the fatty acid profile,namely an increase in oleic acid is more pronounced when YL DGATs areco-expressed with LPAAT-like genes from soy and catalpa.

TABLE 46 Oil concentrations and fatty acid profiles for events generatedby co- transformation with KS387 and pKR561-SOY LPAAT-like KS387 area %pKR561-SOY LPAAT-like % 16:0 18:0 18:1 18:2 18:3 oil  1 11.1 6.4 35.439.9 7.3 13.4  2 12.5 5.4 30.6 43.2 8.4 13.2  3 11.3 6.0 37.6 39.1 6.112.6  4 12.2 6.5 32.4 41.3 7.6 12.4  5 11.3 9.4 39.8 32.8 6.7 12.0  610.6 5.6 34.0 41.5 8.3 11.8  7 14.2 6.1 23.2 46.3 10.1 11.5  8 10.9 5.534.3 41.1 8.3 11.5  9 11.0 5.2 37.2 38.9 7.7 11.1 10 12.0 6.8 33.3 39.68.3 11.1 11 14.4 4.8 22.4 47.0 11.4 10.7 12 11.2 6.0 37.6 37.4 7.8 9.513 11.5 4.9 31.4 41.5 10.7 9.4 14 12.3 5.6 31.9 39.9 10.3 8.6 15 11.65.3 32.3 40.7 10.1 8.4 16 15.3 6.4 23.8 44.0 10.5 7.6 17 14.7 6.1 23.743.7 11.8 7.4 18 14.4 7.2 18.7 43.8 15.9 7.3 19 13.8 5.4 20.0 45.2 15.57.1 20 14.5 7.3 29.5 38.3 10.4 6.9 21 17.1 5.9 22.3 39.8 14.8 6.9 2213.3 4.0 18.0 48.3 16.4 6.2 23 12.4 4.7 24.8 44.6 13.6 6.1 24 13.3 5.028.0 40.3 13.4 6.0 25 15.3 4.9 19.6 44.3 15.9 6.0 26 13.8 4.7 22.7 44.314.5 5.8 27 13.9 4.6 17.1 47.8 16.5 5.7 28 15.6 6.7 25.0 38.6 14.2 5.629 15.5 6.7 25.6 41.1 11.1 5.4 30 14.5 4.8 16.7 46.7 17.2 5.0 average13.2 5.8 27.6 42.0 11.4 8.7

TABLE 48 Oil concentrations and fatty acid profiles for events generatedby co- transformation with KS387 and pKR561-CATALPA LPAAT-like KS387area % pKR561-CATALPA LPAAT-like % 16:0 18:0 18:1 18:2 18:3 oil  1 10.75.7 32.4 44.0 7.2 14.5  2 10.5 6.8 37.2 38.9 6.6 12.8  3 11.9 5.9 37.837.0 7.3 10.6  4 11.7 5.8 31.4 42.3 8.8 10.5  5 11.5 7.0 33.6 39.8 8.210.1  6 12.3 6.8 37.4 35.3 8.2 10.0  7 14.2 6.2 25.7 43.1 10.9 9.4  813.2 5.3 28.9 42.3 10.2 9.3  9 13.4 6.7 29.0 41.1 9.9 9.2 10 12.2 5.728.8 42.3 11.0 9.1 11 12.9 6.7 30.4 41.2 8.8 9.1 12 11.1 5.6 32.0 37.214.2 9.0 13 13.2 5.8 28.0 42.4 10.5 8.9 14 13.1 5.3 27.1 43.7 10.7 8.915 13.1 7.9 29.3 40.8 8.9 8.8 16 10.8 5.6 34.2 39.8 9.6 8.5 17 15.9 5.421.2 46.0 11.4 8.4 18 10.2 6.2 36.2 37.7 9.8 8.4 19 14.3 7.5 28.6 38.611.0 7.2 20 13.2 5.6 25.1 41.6 14.5 6.6 21 14.8 5.7 22.7 43.5 13.3 6.622 12.6 5.4 26.8 41.6 13.7 6.3 23 13.7 7.3 26.3 40.0 12.7 6.2 24 15.65.2 16.3 47.3 15.7 5.8 25 16.4 4.8 16.9 45.3 16.6 5.3 26 15.1 5.4 19.443.6 16.4 5.0 27 14.2 5.5 18.6 43.3 18.3 4.5 average 13.0 6.0 28.2 41.511.3 8.5

TABLE 49 Oil concentrations and fatty acid profiles for events generatedby transformation with KS387 KS387 area % % 16:0 18:0 18:1 18:2 18:3 oil 1 11.3 7.7 31.6 41.4 8.0 13.3  2 11.5 6.9 33.4 40.8 7.4 12.8  3 11.66.0 31.6 42.7 8.1 11.3  4 11.1 6.1 31.7 43.0 8.2 10.8  5 13.1 6.5 27.543.6 9.4 10.6  6 12.8 7.4 28.7 42.5 8.6 10.5  7 15.1 5.3 21.4 46.9 11.39.3  8 16.6 5.4 17.7 48.7 11.7 8.7  9 10.0 6.8 35.6 38.6 9.0 8.6 10 11.46.9 32.9 40.1 8.7 8.1 11 15.7 6.2 19.7 45.7 12.8 7.8 12 13.4 6.6 29.040.8 10.3 7.7 13 15.0 6.4 21.8 45.2 11.7 7.5 14 16.9 6.2 22.2 42.0 12.87.3 15 14.9 6.7 21.3 44.1 13.0 7.0 16 15.2 6.9 20.7 43.8 13.3 7.0 1715.6 5.8 19.1 45.2 14.3 6.9 18 15.1 6.1 20.4 45.1 13.2 6.9 19 15.6 6.320.6 45.0 12.5 6.9 20 16.2 6.0 18.3 45.9 13.6 6.7 21 15.1 6.1 22.0 43.413.4 6.4 22 12.4 5.6 27.4 41.8 12.9 6.4 23 14.2 5.8 18.4 43.9 17.8 6.124 15.7 5.4 18.3 45.0 15.5 5.8 25 16.0 5.9 18.1 45.2 14.8 5.6 26 15.65.6 18.8 44.4 15.6 5.3 27 12.8 4.4 13.4 48.2 21.2 5.2 average 14.1 6.223.8 43.8 12.2 8.0

Example 24 Co-Expression of a LPAAT-Like Gene from Soybean with YL ACBPand YL DGAT1 YL DGAT2 in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1358, comprising a LPAAT-like gene from soybean and pKR1364,comprising a LPAAT-like gene from soybean and YL ACBP and theco-transformation of these vectors with pKR1236, comprising YarrowiaDGAT1 and DGAT2, into somatic embryos. Vector pKR1236 alone was alsoincluded as a control.

Construction of pKR1358 Comprising a Soy LPAAT-Like Gene

The nucleotide and amino acid sequences for a soy LPAAT-like gene areset forth in SEQ ID NO:155 and SEQ ID NO:156, respectively. The BsiWIfragment of pKR561-SOY LPAAT-like (SEQ ID NO:159), was cloned into theBsiWI site of pKR268 (previously described in PCT Publication No. WO04/071467) to produce pKR1358 (SEQ ID NO:170).

Construction of pKR1364 Comprising a Soy LPAAT-Like Gene and YL ACBP

The PstI fragment of pKR1358 (SEQ ID NO:170), containing a soyLPAAT-like gene was cloned into the SbfI site of pKR1250 (SEQ ID NO:118;Example 20) to produce pKR1364 (SEQ ID NO:171).

Co-Expression in Soybean Somatic Embryos of Either YL ACBP, a SoyLPAAT-Like Gene or YL ACBP and a Soy LPAAT-Like Gene with YL DGAT1 andYL DGAT2

Vector pKR1250 (SEQ ID NO:118) and pKR1358 (SEQ ID NO:170) were digestedwith BsiWI and fragments were purified as described in Example 5.Fragment from pKR1364 (SEQ ID NO:171) was similarly prepared afterdigestion with Acc651/FspI. Soybean embryogenic suspension culture (cv.Jack) was transformed with the BsiWI fragment from pKR1250 (SEQ IDNO:118) and pKR1236 (SEQ ID NO:75) and having experiment number MSE2354or with the BsiWI fragment of pKR1358 (SEQ ID NO:170) and pKR1236 (SEQID NO:75) and having experiment number MSE2355 or with the Acc651/FspIfragment of pKR1364 (SEQ ID NO:171) and pKR1236 (SEQ ID NO:75) andhaving experiment number MSE2356. Plasmid pKR1236 (SEQ ID NO:75) wasalso transformed alone in a similar way for a control and has experimentnumber MSE2353.

Example 25 Cloning of Kennedy Pathway Genes from Yarrowia lipolytica

The present example describes cloning of Kennedy pathway genes fromYarrowia. Genes described here were co-transformed with YL DGAT genesinto soybean somatic embryos as described in Examples 18-20.

pFBAIn-YLPAT1

Y. lipolytica LPAT1 cDNA was cloned by amplification of the ORF usingprimers YLPAT1-F (SEQ ID NO:172; GATGATCCATGGTGGTCGTGGACGATGTTC) andYLPAT1-R (SEQ ID NO:173; GATCGAGCGGCCGCTGCAGTTAGTTAGTATCACTTATC). Thereaction mixture contained 1 μl of each primers at 20 μM, 1 μl Y.lipolytica cDNA as template, 10 μl 5×HF buffer for Phusion polymerase(New England Biolab), 1 μl dNTP mix (10 mM each) 34 μl water and 1 μlPhusion polymerase. Amplification condition was as follows: initialdenaturation at 98° C. for 60 sec, followed by 30 cycles of denaturationat 98° C. for 10 sec, annealing at 55° C. for 10 sec, and elongation at72° C. for 30 sec. A final elongation cycle at 72° C. for 3 min wascarried out, followed by reaction termination at 4° C. A ˜690 bp DNAfragment was obtained. This fragment was purified with Qiagen PCRpurification kit according to the manufacturer's protocol. The purifiedPCR product was digested with NcoI and NotI and cloned into pFBAIn-MOD-1(SEQ ID NO:18) between NcoI and NotI. The resulting plasmid was namedpFBAIn-YLPAT1 (SEQ ID NO:104).

pFBAIn-YLPAAT2

Y. lipolytica LPAT2 cDNA was cloned by amplification of the ORF usingprimers YLPAAT2-FNCOI (SEQ ID NO:174; ACGTCCATGGCCGTTGCATCCAAGCTCGT) andYLPAAT2-RNOTI (SEQ ID NO:175; ACGTGCGGCCGCCTACTGAGTCTTCTGGCCAGCGT). Thereaction mixture contained 0.5 μl of the cDNA, 1 μl each of the primersat 20 μM, 22 μl water and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRaBio Inc., Otsu, Shiga, 520-2193, Japan). Amplification was carried outas follows: initial denaturation at 94° C. for 60 sec, followed by 30cycles of denaturation at 94° C. for 20 sec, annealing at 55° C. for 20sec, and elongation at 72° C. for 60 sec. A final elongation cycle at72° C. for 3 min was carried out, followed by reaction termination at 4°C. A ˜850 bp DNA fragment was obtained. This fragment was purified withQiagen PCR purification kit according to the manufacturer's protocol.The purified PCR product was digested with NcoI and NotI and cloned intopFBAIn-MOD-1 (SEQ ID NO:18) between NcoI and NotI. The resulting plasmidwas named pFBAIn-YLPAAT2) SEQ ID NO:108).

pFBAIn-YLPAT3

Y. lipolytica LPAT3 cDNA was cloned by amplification of the ORF usingprimers YLPAT3-F (SEQ ID NO:176; GAAGATCCATGGCTACTGCAGAGGCTGTCAAG) andYLPAT3-R (SEQ ID NO:177; GAAGATCGCGGCCGCCTTACTAACAACTTCAGATCGTC). Thereaction mixture contained 1 μl of each primers at 20 μM, 1 μl Y.lipolytica cDNA as template, 10 μl 5×HF buffer for Phusion polymerase(New England Biolab), 1 μl dNTP mix (10 mM each), 34 μl water and 1 μlPhusion polymerase. Amplification condition was as follows: initialdenaturation at 98° C. for 60 sec, followed by 30 cycles of denaturationat 98° C. for 10 sec, annealing at 55° C. for 10 sec, and elongation at72° C. for 40 sec. A final elongation cycle at 72° C. for 3 min wascarried out, followed by reaction termination at 4° C. A ˜1250 bp DNAfragment was obtained. This fragment was purified with a Qiagen PCRpurification kit according to the manufacturer's protocol. The purifiedPCR product was digested with NcoI and NotI and cloned into pFBAIn-MOD-1(SEQ ID NO:18) between NcoI and NotI. The resulting plasmid was namedpFBAIn-YLPAT3 (SEQ ID NO:111).

pYAT-ACBP

The Y. lipolytica ACBP cDNA was cloned as follows. Primers ACBP-F1 (SEQID NO:178; GATCAACCATGGCTTCCGCCGAATTCACCGCCGCTGCCGACTC) and ACBP-R (SEQID NO:179; GATCAAGCGGCCGCTTAGTTGTACTTGTCGGAAAG) were used to amplify theACBP ORF from the cDNA of Y. lipolytica by PCR. The reaction mixturecontained 0.5 μl of the cDNA, 1 μl each of the primers at 20 μM, 22 μlwater and 25 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc., Otsu,Shiga, 520-2193, Japan). Amplification was carried out as follows:initial denaturation at 94° C. for 60 sec, followed by 30 cycles ofdenaturation at 94° C. for 20 sec, annealing at 55° C. for 20 sec, andelongation at 72° C. for 20 sec. A final elongation cycle at 72° C. for3 min was carried out, followed by reaction termination at 4° C. A ˜250bp DNA fragment was obtained from the PCR reaction. It was purifiedusing Qiagen's PCR purification kit according to the manufacturer'sprotocol. The purified PCR product was digested with NcoI and NotI, andcloned into Nco I-Not I digested pYAT-MOD-1 vector (SEQ ID NO:180), suchthat the gene was under the control of the Y. lipolytica YAT1 promoter(PCT Publication No. WO 2006/052754) and the PEX20-3′ terminator region.Correct transformants were confirmed by miniprep analysis and theresultant plasmid was designated as pYAT-ACBP (SEQ ID NO:116). Thenucleotide sequence of the YL ACBP gene comprised in pYAT-ACBP andcorresponding amino acid sequence of the YL ACBP protein are set forthas SEQ ID NO:181 and SEQ ID NO:182, respectively.

pFBAIn-YCPT1

The Y. lipolytica CPT1 cDNA was cloned as follows. Primers CPT1-5′-NcoI(SEQ ID NO:183; ACGTCCATGGGCGTATTCATTAAACAGG) and CPT1-3′-NotI (SEQ IDNO:184; ACGTGCGGCCGCTTATTCGGTCTTCTTCTCCTG) were used to amplify the Y.lipolytica ORF from the cDNA of Y. lipolytica by PCR. The reactionmixture contained 0.5 μl of the cDNA, 0.5 μl each of the primers, 11 μlwater and 12.5 μl ExTaq premix 2×Taq PCR solution (TaKaRa Bio Inc.,Otsu, Shiga, 520-2193, Japan). Amplification was carried out as follows:initial denaturation at 94° C. for 300 sec, followed by 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, andelongation at 72° C. for 60 sec. A final elongation cycle at 72° C. for10 min was carried out, followed by reaction termination at 4° C. A˜1190 bp DNA fragment was obtained from the PCR reaction. It waspurified using Qiagen's PCR purification kit according to themanufacturer's protocol. The purified PCR product was digested with NcoIand NotI, and cloned into Nco I-Not I digested pFBAIn-MOD-1 vector (SEQID NO:18; described in PCT Publication No. WO2007/061742), such that thegene was under the control of the Y. lipolytica FBAIN promoter (U.S.Pat. No. 7,202,356) and the PEX20Pex20 terminator sequence of theYarrowia Pex20 gene (GenBank Accession No. AF054613) (i.e., PEX20-3′).Correct transformants were confirmed by miniprep analysis and theresultant plasmid was designated as pFBAIn-YCPT1 (SEQ ID NO:119).

TABLE 50 Nucleotide sequences and corresponding amino acid sequences ofYarrowia kennedy pathway genes comprised in soybean transformationvectors Vector name/ Kennedy pathway SEQ ID NO: SEQ ID NO: SEQ ID NO:gene DNA sequence AA sequence pKR1239/105 YL LPAAT1 185 186 pKR1243/110YL LPAAT2 187 188 pKR1244/113 YL LPAAT3 189 190 pKR1246/115 YL PDAT 191192 pKR1251/121 YL CPT 193 194

Example 26 Co-Expression of YL DGAT1 with a FAD2/TE2 Down RegulationConstruct in Soybean Somatic Embryos

The present example describes construction of soybean expression vectorspKR1274, comprising Yarrowia DGAT1 (YL DGAT1) and either pKR1267 orpKR1269, comprising a soybean fatty acid desaturase 2 (GMFAD2)/thioesterase 2 (GM TE2) down-regulation construct. While the GMFAD2-TE2 down-regulation region of pKR1267 and pKR1269 are identical ineach construct and both are driven by the KTi3 promoter, pKR1267contains only the KTi3 terminator and pKR1269 contains both the KTi3 andsoy albumin terminators.

Construction of pKR1274 comprising YL DGAT1

A starting plasmid pKR85 (SEQ ID NO:27), which was previously describedin Example 4 contains the hygromycin B phosphotransferase gene (HPT)(Gritz, L. and Davies, J., Gene 25:179-188 (1983)), flanked by the T7promoter and transcription terminator (T7prom/hpt/T7term cassette), anda bacterial origin of replication (ori) for selection and replication inbacteria (e.g., E. coli). In addition, pKR72 also contains thehygromycin B phosphotransferase gene, flanked by the 35S promoter (Odellet al., Nature 313:810-812 (1985)) and NOS3′ transcription terminator(Depicker et al., J. Mol. Appl. Genet. 1:561-570 (1982)) (35S/hpt/NOS3′cassette) for selection in plants such as soybean. Plasmid pKR85 (SEQ IDNO:27) also contains a NotI restriction site, flanked by the promoterfor the a′ subunit of β-conglycinin (Beachy et al., EMBO J. 4:3047-3053(1985)) and the 3′ transcription termination region of the phaseolingene (Doyle et al., J. Biol. Chem. 261:9228-9238 (1986)), calledBcon/NotI/Phas3′ cassette.

The Bcon/NotI/Phas3′ cassette was removed from pKR85 (SEQ ID NO:27) bydigestion with HindIII and the resulting fragment was re-ligated toproduce pKR278 (SEQ ID NO:195).

The BsiWI fragment of pKR1235 (SEQ ID NO:74, Example 14), containing theYL DGAT1, was cloned into the BsiWI site of pKR278 (SEQ ID NO:195),which was previously described in U Pat. Pub. US20080095915 (thecontents of which are incorporated by reference), to produce pKR1274(SEQ ID NO:196).

Construction of pKR1267 Comprising GM FAD2-TE2 Down-Regulation Cassette

The 5′ end of GM TE2 (SEQ ID NO:197) was amplified from pTC4 (SEQ IDNO:198), which was previously described in WO1996006936Δ1 (the contentsof which are incorporated by reference), with oligonucleotide primersGmTE2_5-1 (SEQ ID NO:199) and GmTE2_3-1 (SEQ ID NO:200), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The 3′ end of GM TE2(SEQ ID NO:197) was amplified from pTC4 (SEQ ID NO:198) witholigonucleotide primers GmTE2_5-2 (SEQ ID NO:201) and GmTE2_3-2 (SEQ IDNO:202), using the Phusion™ High-Fidelity DNA Polymerase (Cat. No.F553S, Finnzymes Oy, Finland) following the manufacturer's protocol. Theresulting two PCR products were combined and amplified with GmTE2 5-1(SEQ ID NO:199) and GmTE2_3-2 (SEQ ID NO:202) using the Phusion™High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy, Finland)following the manufacturer's protocol. The resulting DNA fragment wascloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCRCloning Kit (Invitrogen Corporation), following the manufacturer'sprotocol, to produce pKR1258 (SEQ ID NO:203).

The 5′ end of GM FAD2 (SEQ ID NO:204) was amplified from pBS43 (SEQ IDNO:205), which was previously described in WO1997047731Δ2 (the contentsof which are incorporated by reference), with oligonucleotide primersGmFAD2-1_5-1 (SEQ ID NO:206) and GmFAD2-1_3-1 (SEQ ID NO:207), using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The 3′ end of GM FAD2-1(SEQ ID NO:204) was amplified from pBS43 (SEQ ID NO:205) witholigonucleotide primers GmFAD2-1_5-2 (SEQ ID NO:208) and GmFAD2-1_3-2(SEQ ID NO:209), using the Phusion™ High-Fidelity DNA Polymerase (Cat.No. F553S, Finnzymes Oy, Finland) following the manufacturer's protocol.The resulting two PCR products were combined and amplified withGmFAD2-1_5-1 (SEQ ID NO:206) and GmFAD2-1_3-2 (SEQ ID NO:209) using thePhusion™ High-Fidelity DNA Polymerase (Cat. No. F553S, Finnzymes Oy,Finland) following the manufacturer's protocol. The resulting DNAfragment was cloned into the pCR-Blunt® cloning vector using the ZeroBlunt® PCR Cloning Kit (Invitrogen Corporation), following themanufacturer's protocol, to produce PCRblunt-Fad2-1 (SEQ ID NO:210).

The MluI fragment of pKR1258 (SEQ ID NO:331), containing GM TE2, wascloned into the MluI fragment of PCRblunt-Fad2-1 (SEQ ID NO:210),containing GM FAD2-1, to produce pKR1259 (SEQ ID NO:211).

The EcoRI fragment of pKR1259 (SEQ ID NO:211) comprised of the 5′ end ofthe GM FAD2/TE2 fragment, was cloned into the MfeI site of pKR1259 (SEQID NO:211) to produce pKR1261(SEQ ID NO:212) which contains a GMFAD2-TE2-TE2loop-TE2-FAD2 hairpin structure flanked by NotI sites.

The NotI fragment of pKR1261(SEQ ID NO:212), containing GMFAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR123R (SEQID NO:213), which was previously described in WO2004071467Δ2 (thecontents of which are incorporated by reference), to produce pKR1266(SEQ ID NO:214).

The BsiWI/PstI fragment of pKR1266 (SEQ ID NO:214), containing the GMFAD2-TE2-TE2loop-TE2-FAD2 was cloned into the BsiWI/SbfI fragment ofpKR278 (SEQ ID NO:195) to produce pKR1267 (SEQ ID NO:215).

Construction of pKR1269 Comprising GM FAD2-TE2 Down-Regulation Cassette

The NotI fragment of pKR1261(SEQ ID NO:212), containing GMFAD2-TE2-TE2loop-TE2-FAD2, was cloned into the NotI site of pKR457 (SEQID NO:216), which was previously described in PCT Publication No. WO2005/047479 (the contents of which are hereby incorporated byreference), to produce pKR1264 (SEQ ID NO:1264).

The PstI fragment of pKR1264 (SEQ ID NO:217), containing the GMFAD2-TE2-TE2loop-TE2-FAD2 was cloned into the SbfI fragment of pKR277(SEQ ID NO:218), which was previously described in PCT Publication No.WO 2004/071467 to produce pKR1269 (SEQ ID NO:219).

Co-Expression of GM FAD2-TE2-TE2loop-TE2-FAD2 Down-Regulation ConstructsEither Alone or with YL DGAT2

Soybean embryogenic suspension culture (cv. Jack) was transformed withthe pKR1267 (SEQ ID NO:215) alone and having experiment number MSE2213or with the BsiWI fragment of pKR1269 (SEQ ID NO:219) and pKR1274 (SEQID NO:196) and having experiment number MSE2210. Events were selectedand somatic embryos matured in SHaM as described in Example 5. Oilconcentrations and fatty acid profiles were determined as described inExample 4 for MSE2213 and MSE2210 and results for each experiment areshown in Table 51 and Table 52, respectively.

TABLE 51 Oil concentrations and fatty acid profiles for events fromMSE2213 MSE2213 (GM FAD2-TE2-TE2loop-TE2-FAD2) % Event 16:0 18:0 18:118:2 18:3 Oil 2213-16 5.8 2.6 72.7 10.5 8.5 16.8 2213-26 12.0 5.7 24.847.2 10.4 16.5 2213-30 11.7 5.3 18.5 53.1 11.4 15.4 2213-23 9.0 3.2 52.224.7 11.0 15.3 2213-29 13.1 3.7 13.3 57.1 12.8 15.0 2213-7 6.4 3.1 66.315.6 8.6 14.9 2213-31 11.7 3.0 26.9 46.5 11.9 14.5 2213-13 6.7 3.3 64.516.6 8.9 14.4 2213-5 3.5 3.0 78.0 7.5 8.1 13.8 2213-9 7.6 3.4 57.6 20.311.0 13.2 2213-20 7.8 4.1 56.7 20.8 10.6 13.1 2213-4 4.1 2.7 72.1 11.110.0 12.7 2213-17 7.7 5.2 57.0 19.7 10.4 12.5 2213-3 7.4 3.6 64.4 14.410.2 12.5 2213-24 12.9 7.3 29.4 39.0 11.4 12.1 2213-1 13.8 7.1 22.1 44.612.4 11.9 2213-6 6.7 2.2 57.2 22.2 11.7 11.8 2213-11 9.2 5.2 58.2 16.411.0 10.9 2213-2 7.8 4.3 45.3 31.0 11.7 10.6 2213-14 7.3 4.6 63.0 15.010.2 10.5 2213-27 8.5 6.0 48.9 25.0 11.6 10.1 2213-19 8.0 4.0 53.7 21.412.8 9.9 2213-21 11.1 5.6 28.3 40.6 14.4 9.8 2213-18 7.4 4.1 57.2 17.713.5 9.4 2213-12 6.4 4.3 63.2 15.5 10.6 9.4 2213-10 13.8 7.0 24.0 42.113.2 9.4 2213-28 8.8 4.1 54.1 19.1 13.8 9.1 2213-25 6.9 3.5 53.9 21.913.8 9.0 2213-15 5.2 3.4 61.7 17.6 12.1 8.0 2213-8 14.4 6.6 20.0 42.916.1 7.6 2213-22 14.3 6.3 21.3 41.9 16.2 7.2 Avg. 8.9 4.4 47.9 27.1 11.611.9

TABLE 52 Oil concentrations and fatty acid profiles for events fromMSE2210 MSE2210 (YL DGAT1 & GM FAD2-TE2-TE2loop-TE2-FAD2) % Event 16:018:0 18:1 18:2 18:3 Oil 2210-2 3.8 2.7 74.7 13.1 5.8 22.1 2210-23 10.36.8 32.0 44.4 6.5 20.0 2210-29 7.5 5.5 63.9 17.1 6.0 20.0 2210-19 3.93.5 79.1 8.1 5.4 19.7 2210-10 7.3 3.8 66.6 15.6 6.7 19.6 2210-12 5.7 3.667.6 17.6 5.6 19.6 2210-14 4.5 2.4 67.9 17.8 7.4 19.3 2210-25 5.1 3.571.8 13.1 6.4 18.4 2210-5 9.0 4.1 43.1 35.9 7.9 17.8 2210-24 13.7 4.320.5 52.5 9.1 17.5 2210-13 10.2 5.5 36.8 40.4 7.0 17.4 2210-11 10.9 7.830.6 43.2 7.5 16.6 2210-1 4.1 3.0 75.6 9.6 7.7 15.9 2210-6 2.7 1.7 83.95.3 6.4 15.5 2210-16 8.4 3.7 48.5 31.3 8.1 15.3 2210-26 7.1 4.9 55.224.7 8.1 14.4 2210-7 4.3 3.2 62.9 20.8 8.7 14.3 2210-20 6.8 4.3 65.215.7 7.9 13.8 2210-27 10.9 7.4 42.4 30.0 9.3 13.8 2210-30 5.0 2.4 65.117.1 10.4 13.7 2210-3 7.1 4.8 52.9 27.4 7.9 13.6 2210-4 6.0 3.7 66.015.9 8.5 13.3 2210-22 3.2 3.3 77.7 8.1 7.8 13.2 2210-8 9.1 4.9 49.7 27.39.0 13.1 2210-21 6.0 3.5 67.8 14.9 7.8 12.8 2210-18 12.7 5.2 20.2 49.712.1 12.5 2210-31 4.4 2.9 73.1 10.1 9.5 11.7 2210-9 4.0 3.1 74.1 9.3 9.611.2 2210-15 3.5 2.4 72.5 11.0 10.6 11.1 2210-28 14.1 5.7 20.2 45.5 14.510.1 2217-29 12.0 7.5 28.0 38.8 13.8 8.7 Avg. 7.2 4.2 56.6 23.6 8.4 15.4

Comparison of results in Tables 51 and 52 demonstrates that combinationof YL DGAT1 expression with down-regulation of GM FAD2-1 and GM TE2changes the fatty acid profile to an extend that exceeds the change thatobserved when only GM FAD2-1 and GM TE2 genes are suppressed.

Example 27 Construction of Chimeric Vectors for Seed-Targeted Silencingof Galactinol Synthase and Plastidial Phosphoglucomutase.

Amplification of Partial Galactinol Synthase Polynucleotides:Polynucleotide fragments encoding parts of the galactinol synthase 1(GAS1, described in Applicants' Assignee's U.S. Pat. No. 5,648,210;Issued Jul. 15, 1997; Attorney Docket No. BB1032USPCT), galactinolsynthase 2 (GAS2; Applicants' Assignee's U.S. Pat. No. 6,967,262; IssuedNov. 22, 2005; Attorney Docket No. BB1442USPCT) and galactinol synthase3 (GAS3; described in Applicants' Assignee's U.S. Pat. No. 7,294,756 B2;Issued Nov. 13, 2007; Attorney Docket No. BB1539USNA) were amplified bystandard PCR methods using Pfu Turbo DNA polymerase (Stratagene, LaJolla, Calif.) and the following primer sets. The DNA template for thePCR reaction was plasmid SH58 (SEQ ID NO: 220).

Forward primer G1HPFW04 forward primer (SEQ ID NO: 221):aagcttgcggccgctagtcgactaagtcatcaactattccaagctacReverse primer G3HPRV02 reverse primer (SEQ ID NO: 222):aagcttcgtacgcctaggctacttccccgtatatctccatggcttggThe oligonucleotide primers were designed to add Not I and SalIrestriction endonuclease sites at the 5′ end of GAS1 and AvrII and BsiWIsites to the 3′ end of GAS3. The DNA sequence comprising the resultingpolynucleotide (H-Not1-SalI-GAS1sGAS2GAS3-AvrII-B-H) is shown in SEQ IDNO:223.

Preparation of PHP25069:

The polynucleotide product obtained from the amplification describedabove (H-NotI-Sal1-GAS1sGAS2GAS3-AvrII-B-H, SEQ ID NO:223) was digestedwith SalI and AvrII and assembled into vector pJMS33 (SEQ ID NO: 224,described in U.S. application Ser. No. 11/133,075 filed May 19, 2005) bythe following steps. First, the plasmid pJMS33 was digested with Sal1and AvrII. Then, the isolated DNA fragment containing partial sequencesof GAS1, GAS2 and GAS3 was inserted into Sal1/AvrlII-digested plasmidpJMS33 to obtain an intermediary vector. Secondly, the polynucleotideproduct obtained from the amplification described above(H-NotI-Sal1-GAS1sGAS2GAS3-AvrII-B-H, SEQ ID NO:223) was digested withNotI and BsiWI and assembled into the NotI/BiWI-digested intermediaryvector to obtain plasmid PHP25069 (SEQ ID NO: 225).

Amplification of Partial Plastidial Phosphoglucmutase (pPGM):

A polynucleotide fragment encoding a part of pPGM (pPGM is described inApplicants' Assignee's U.S. Pat. No. 7,250,557; Issued Jul. 31, 2007;Attorney Docket No. BB1451USNA), was amplified by standard PCR methodsusing Pfu Turbo DNA polymerase (Stratagene, La Jolla, Calif.) and thefollowing primer sets. The DNA template for the PCR reaction was plasmidpTC103 (described in Applicants' Assignee's U.S. Pat. No. 7,250,557;Issued Jul. 31, 2007; Attorney Docket No. BB1451USNA).

Forward primer GMPGMFW02 primer (SEQ ID NO: 226):gaattccctaggtgagctgatttaagatttatcaaaagttgReverse primer GMPGMRV02 primer (SEQ ID NO: 227):GatatccctaggcgtacgttaaatcagctcagaaagggaggttcThe oligonucleotide primers were designed to add an AvrII restrictionendonuclease site at the 3′ and at the 5′ end of the pPGM fragment. TheDNA sequence comprising the resulting polynucleotide (PGMPCRAA) is shownin SEQ ID NO:228.

Preparation of PHP28656:

The polynucleotide product PGMPCRAA (SEQ ID NO:228) was digested withAvrII and assembled into an AvrII-digested vector PHP25069 (SEQ ID NO:225) to obtain vector PHP28656 (SEQ ID NO: 229).

Preparation of PHP29252:

The polynucleotide product of SEQ ID NO:223 was digested with SalI andAvrII and assembled into vector pJMS33 (SEQ ID NO: 224) to obtain anintermediary vector SH73.

A polynucleotide fragment representing a potato intron (STLS1) wasamplified by standard PCR methods using Pfu Turbo DNA polymerase(Stratagene, La Jolla, Calif.) and the following primer sets. The DNAtemplate for the PCR reaction was plasmid PHP21155 (SEQ ID NO:230).

Forward primer PHP21155FW01 (SEQ ID NO: 231):gaattccctgcaggacctgcacatcaccatgttttggtcaReverse primer PHP21155RV01 (SEQ ID NO: 232)gaattccgtacgcaggtaagtttctgcttctacctttgThe DNA sequence comprising the resulting polynucleotide(STLS1-BsiWI/SbfI) is shown in SEQ ID NO:233.

A polynucleotide fragment encoding a part of pPGM was amplified bystandard PCR methods using Pfu Turbo DNA polymerase (Stratagene, LaJolla, Calif.) and a primer set designed to add an AvrII restrictionendonuclease site at the 3′ of the pPGM fragment and a SbfI, PstI andEcoRV sites at the 5′ end of the pPGM fragment. The DNA sequencecomprising the resulting polynucleotide (PGMPCRAS) is shown in SEQ IDNO:234.

A second intermediary vector, pJMS40, was assembled by ligating theBsiWI/SbfI-digested STLS1 intron (SEQ ID NO:233) and the AvrII/SbfIdigested PGMPCRAS (SEQ ID NO:234) into the AvrII/BsiWI digested vectorSH73.

PHP29252 (SEQ ID NO: 235) was assembled by ligating a 2168 bp Not1-BsiWIfragment of PHP28656 (SEQ ID NO: 229), containing the partial GAS1 GAS2

GAS3 polynucleotides linked to the partial pPGM polynucleotide, into theNotI/BsiWI-digested pJMS40 (containing the STLS1 intron linked to theinverse partial GAS1 GAS2 GAS3 pPGM polynucleotides). PHP29252 canencode for a hairpin structure with the partial GAS1 GAS2 GAS3 pPGMpolynucleotides representing the stem structure of the hairpin and theSTLS1 intron representing the loop structure.

Example 28 Transformation of Soybeans with Chimeric Vectors forSeed-Targeted Silencing of Galactinol Synthase and PlastidialPhosphoglucomutase

Soybean were transformed with a seed-specific expression AscI fragmentof PHP29252 (SEQ ID NO: 235) containing the KTi promoter linked to theGAS1 GAS2 GAS3 PGM hairpin structure described in Example 27 by themethod of particle gun bombardment (Klein, T. M. et al. (1987) Nature(London) 327:70-73; U.S. Pat. No. 4,945,050; described in Example 6).

The AscI-PHP29252 fragment was co-bombarded with a DNA fragment(PHP19031A, SEQ ID NO: 236) containing the ALS selectable marker drivenby the soybean SAMS promoter.

Soybean somatic embryos as well as mature seeds were analyzed asdescribed in Example 29 and 30

Example 29 Raffinose Family Oligosaccharide (RFO) Analysis in TransgenicSoybean Somatic Embryos and Soybean Seeds

Individual immature soybean embryos were dried-down (by transferringthem into an empty small Petri dish that was seated on top of a 10 cmPetri dish containing some agar gel to allow slow dry down) to mimic thelast stages of soybean seed development. Dried-down embryos are capableof producing plants when transferred to soil or soil-less media. Storageproducts produced by embryos at this stage are similar in composition tostorage products produced by zygotic embryos at a similar stage ofdevelopment and most importantly the storage product profile ispredictive of plants derived from a somatic embryo line (PCT PublicationNo. WO 94/11516, which published on May 26, 1994). Raffinose FamilyOligosaccharides (raffinose, stachyose) of transgenic somatic embryoscontaining recombinant expression construct described in Example 27 wasmeasured by thin layer chromatography. Somatic embryos were extractedwith hexane then dried. The dried material was re-suspended in 80%methanol, incubated at room temperature for 1-2 hours, centrifuged, and2 μl of the supernatant is spotted onto a TLC plate (Kieselgel 60 CF,from EM Scientific, Gibbstown, N.J.; Catalog No. 13749-6). The TLC wasrun in ethylacetate:isopropanol:20% acetic acid (3:4:4) for 1-1.5 hours.The air dried plates were sprayed with 2% sulfuric acid and heated untilthe charred sugars were detected. The somatic embryos from event4871.6.10 showed reduced levels of raffinose sugars (raffinose andstachyose) when compared to untransformed wild type soybean (WT) somaticembryos.

Mature soybean T1 and T2 seeds of event 4871.6.10 were chipped and thechips were analyzed by TLC as described above. Seed from events4871.6.10 showed reduced levels of raffinose sugars (raffinose andstachyose) when compared to untransformed wild type soybean (WT) somaticembryos.

Example 30 Down Regulation of Plastidic Phosphoglucomutase in Soybean

Transgenic soybean lines containing the recombinant construct of theinvention were screened for down regulation of plastidicphosphoglucomutase. The screening assay involved iodine staining for thepresence or absence of starch in immature seeds (mid-pod stage) or drieddown embryos (see example 29). The method involved harvesting a slice ofthe immature seed or the entire dried down embryo and placing thistissue in 100% ethanol overnight. Thereafter, the ethanol was decantedand the tissue was subsequently stained with water:lugol (4:1) solutionfor 10 to 30 minutes at room temperature. Lugol is an iodine/potassiumiodide solution, commercially available from Sigma (USA).

The somatic embryos from event 4871.6.10 showed reduced starchaccumulation as indicated by reduced iodine staining.

Example 31 Seed Composition of Event 4871.6.10

T2 seeds derived from a T1 plant that was heterozygous for the 4871.6.10transgenic event were genotyped by TLC analysis of raffinosaccharides.Eight transgenic seeds lacking RFO and eight null segregant seedscontaining RFO were pooled. The soluble carbohydrate and starch levelswere measured as described in Example 13B.

TABLE 53 Composition of T2 seed of event 4871.6.10 g/kg tissue (dry wt);Mean +/− SD of three reps Pini- Sorb- Fruc- Glu- Myo- Suc- Raffi- Stach-Total g/kg tol itol tose cose Inositol rose nose yose (dry wt) StarchNull Mean 1.6 0.4 1.2 1.2 0.2 61.4 9.2 39.2 114.3 0.2 SD 0.0 0.0 0.0 0.20.0 0.9 0.1 1.4 0.4 0.0 TG Mean 3.8 0.1 1.2 1.7 0.3 78.1 1.3 0.2 86.70.1 SD 0.0 0.0 0.1 0.3 0.0 1.7 0.1 0.1 1.6 0.1According to Table 53 seeds of event 4871.6.10 from plants grown in acontrolled environment show a 97% reduction in raffinosaccharides and a27% increase in sucrose. Overall there is a 24% reduction in solublecarbohydrate associated with the presence of the transgene.

Homozygous T3 seeds of event 4871.6.10 and a non-transgenic nullisolines derived from the same event were submitted to EurofinsScientific Inc. (USA) for compositional analysis.

TABLE 54 Composition of T3 seed of event 4871.6.10 % % % % % % % % ProOil sucrose stachyose raffinose crude ash carbs, calc DW DW DW DW DWfiber DW DW DW 4871.6.10 42.4 24.6 6.2 <0.2 <0.2 4.6 8.2 24.8 TG4871.6.10 42.4 23.5 7.8 <0.2 <0.2 4.9 8.2 25.9 TG 4871.6.10 41.2 24.06.6 <0.2 <0.2 5.2 8.3 26.5 TG avg 42.0 24.0 6.9 4.9 8.2 25.7 4871.6.1039.8 22.3 5.1 4.7 1.0 6.1 8.1 29.8 null 4871.6.10 39.6 22.5 4.6 4.4 0.85.6 7.7 30.2 null avg 39.7 22.4 4.9 4.6 0.9 5.9 7.9 30.0According to Table 54 T3 seeds of event 4871.6.10 grown in a controlledenvironment have raffinosaccharide levels below the detection level of0.2% DW and show an increase in oil and protein levels of 1.6 and 2.3%points, respectively and an increase in sucrose levels of 1.7% pointscompared to null segregant seed of the same event.

Homozygous T3 seeds of event 4871.6.10 and a non-transgenic null isolinederived from the same event were planted in three field environments inthe spring of 2008. For each seed source, transgenic or null-segregantseeds of event 4871.6.10, at least three double rows comprised of 220plants and as many as twelve double rows comprised of said number ofplants were planted in a given environment.

TABLE 55 Oil and protein content of T4 seed of event 4871.6.10 grown infield environments % protein % oil 4871.6.10 Null 33.4 18.7 4871.6.10 TG35.9 19.5

Table 55 shows the average oil and protein content as measured by NIRspectroscopy of T4 seed of 4871.6.10 grown in the three different fieldenvironments. There is an increase in oil content of 4% (0.8% points)and an increase in protein content of 7% (2.4% points) associated withthe presence of the 4871.6.10 transgene when plants are grown in a fieldenvironment.

In summary the event 4871.6.10 shows >90% reduction of raffinose andstachyose levels indicative of efficient silencing galactinol synthasegenes (U.S. Pat. No. 5,648,210, U.S. Pat. No. 6,967,262, U.S. Pat. No.7,294,756 B2) and an increase in oil and protein of soybean seedindicative of efficient silencing of plastidal phosglucomutase genes(U.S. Pat. No. 7,250,557).

Example 32 Generation of Soybean Lines with Seed-Targeted Silencing ofGalactinol Synthase and Plastidial Phosphoglucomutase and Seed TargetedOver-Expression of DGAT Enzymes

T2 plants of events EAFS 4818.1.5 and EAFS 4871.6.10 that werehomozygous for an YL DGAT1/YL DGAT2 event (Example 6) and a PGM/RFO koevent (Example 31), respectively were crossed. Resulting F1 seed wereanalyzed by GC and TLC as described above to monitor presence of theDGAT trait (elevated oleic acid) and PGM/RFO ko trait (reduced raffinoseand stachyose). A total of 23 F1 seed with elevated oleic acid contentand reduced raffinosaccharide levels were planted, resulting plants weregrown to maturity. For every F1 plant a total of 48 F2 seed weresubjected to the following analysis sequence. Seed oil content wasmeasured non-destructively by NMR (Example 4). Seed were chipped and theresulting seed chip was extracted with heptane. Heptane extracts weresubjected to GC analysis to determine the presence of the DGAT transgene(indicated by the altered fatty acid profile of the oil). Theheptane-extracted residue was subjected to TLC analysis of raffinose andstachyose content (Example 29) to determine presence or absence of theEAFS 4871.6.10 transgene. In this manner 1104 seed were genotyped andthe effect of transgene genotype on oil content was determined.

TABLE 56 Comparison of oil content of F2 progeny of a cross betweenevents EAFS 4818.1.5 and EAFS 4871.6.10 F1 Δ oil % points plant overnull DGAT + PGM/RFO # PGM/RFO ko DGAT ko null  1 5.3 4.4 2 0  2 4.1 2.70.2 0  3 5.2 4.9 2.2 0  4 3.1 2.2 −0.8 0  5 5.1 4.5 2 0  6 1.8 1.8 −1.90  7 3.8 2.1 0.7 0  8 5.3 3.9 3.3 0  9 5.4 4.2 0.4 0 10 4.9 4.3 0.1 0 114.9 3.6 1 0 12 5 2.7 0.3 0 13 4.5 3.4 1.3 0 14 2.8 2.4 0 0 15 4 3.2 −1.10 16 3.8 2 −1.8 0 17 4.2 4.5 0.9 0 18 3.4 2.8 1.5 0 19 3.9 3.3 −0.2 0 204.8 3.2 1 0 21 5.2 5.6 3 0 22 5.1 4.3 −0.1 0 23 7.5 6.6 2.8 0 avg 4.53.6 0.7 0.0

Table 56 shows that the oil increases associated with the presence of4818.1.5 and 4871.6.10 transgenes are additive. The average oil contentof all F2 seed positive for DGAT and PGM/RFO ko traits is 22.8%. Thereis an oil increase of 4.5% points compared to the average oil content ofall seeds of the same 23 F2 segregants that did not contain DGAT andPGM/RFO ko traits which was 18.3%. Thus combination of DGAT overexpression represented by event 4818.1.5 and plastidal PGM-downregulation represented by event 4871.6.10 allows increasing the oilcontent of soybean seed by more than 20%.

What is claimed is:
 1. A transgenic soybean seed having an increased total fatty acid content of at least 20% and a decreased stachyose content when compared to the total fatty acid content and stachyose content of a non-transgenic, null segregant soybean seed, wherein the seed comprises at least one recombinant construct having at least one diacylglycerol acyltransferase (DGAT) sequence and a construct downregulating galactinol synthase (GAS) activity wherein the DGAT sequence and the GAS construct can be in the same recombinant construct or in separate recombinant constructs.
 2. The transgenic soybean seed of claim 1 further wherein the DGAT sequence is a Yarrowia DGAT sequence.
 3. The transgenic soybean seed of claim 1, wherein the DGAT sequence is a DGAT1.
 4. The transgenic soybean seed of claim 1, wherein the DGAT sequence is a DGAT2.
 5. The transgenic soybean seed of claim 1, wherein the transgenic soybean seed has an increased protein content and decreased raffinose content when compared to the protein content and raffinose content of the non-transgenic, null segregant soybean seed.
 6. The transgenic soybean seed of claim 1, wherein the transgenic soybean seed has a decreased raffinose content when compared to the raffinose content of the non-transgenic, null segregant soybean seed.
 7. The transgenic soybean seed of claim 1, wherein the transgenic soybean seed has an increased total fatty acid content of at least 25% when compared to the total fatty acid content of the non-transgenic, null segregant soybean seed.
 8. A method for increasing the total fatty acid content and decreasing the stachyose content of a soybean seed, the method comprising: (a) transforming at least one soybean cell with at least one recombinant construct having at least one diacylglycerol acyltransferase (DGAT) sequence and a construct downregulating galactinol synthase (GAS) activity wherein the DGAT sequence and the GAS construct can be in the same recombinant construct or in separate recombinant constructs; (b) selecting the transformed soybean cell(s) of step (a) such that plants grown from the transformed cells produce transgenic seed having an increased total fatty acid content of at least 20% and a decreased stachyose content when compared to the total fatty acid content and stachyose content of a non-transgenic, null segregant soybean seed.
 9. The method of claim 8, wherein the DGAT sequence is a DGAT1.
 10. The method of claim 8, wherein the DGAT sequence is a DGAT2.
 11. The method of claim 8, wherein the DGAT sequence is a Yarrowia sequence.
 12. The method of claim 8, wherein the transgenic seed has an increased total fatty acid content of at least 25%.
 13. The method of claim 8, wherein the transgenic soybean seed has increased protein content and decreased raffinose content when compared to the protein content and raffinose content of the non-transgenic, null segregant soybean seed.
 14. The method of claim 8, wherein the the transgenic soybean seed has a decreased raffinose content when compared to the raffinose content of the non-transgenic, null segregant soybean seed.
 15. A meal obtained from a transgenic soybean seed having increased total fatty acid content of at least 20% when compared to the total fatty acid content of a non-transgenic, null segregant soybean seed, wherein the transgenic soybean seed comprises at least one recombinant construct having at least one diacylglycerol acyltransferase (DGAT) sequence and a construct downregulating galactinol synthase (GAS) activity wherein the DGAT sequence and the GAS construct can be in the same recombinant construct or in separate recombinant constructs, further wherein the soluble carbohydrate content of the meal is reduced by at least 17% when compared to the soluble carbohydrate content of a meal obtained from a non-transgenic, null segregant soybean seed.
 16. The meal of claim 15, wherein the DGAT sequence is a Yarrowia DGAT sequence.
 17. The meal of claim 15, wherein the soluble carbohydrate is stachyose.
 18. The meal of claim 15, wherein the soluble carbohydrate is raffinose.
 19. The meal of claim 15, wherein the transgenic soybean seed has an increased total fatty acid content of at least 25%.
 20. The meal of claim 19, wherein the protein content is increased by at least 1.5% points in the meal obtained from the transgenic soybean seed compared to the protein content of a meal obtained from the non-transgenic null segregant soybean seed. 